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Sample records for cd40l dna combined

  1. Multifunctional CD40L: pro- and anti-neoplastic activity.

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    Korniluk, Aleksandra; Kemona, Halina; Dymicka-Piekarska, Violetta

    2014-10-01

    The CD40 ligand is a type I transmembrane protein that belongs to a tumor necrosis factor (TNF) superfamily. It is present not only on the surface of activated CD4+ T cells, B cells, blood platelets, monocytes, and natural killer (NK) cells but also on cancer cells. The receptor for ligand is constitutively expressed on cells, TNF family protein: CD40. The role of the CD40/CD40L pathway in the induction of body immunity, in inflammation, or in hemostasis has been well documented, whereas its involvement in neoplastic disease is still under investigation. CD40L ligand may potentiate apoptosis of tumor cells by activation of nuclear factor-κB (NF-κB), AP-1, CD95, or caspase-depended pathways and stimulate host immunity to defend against cancer. Although CD40L has a major contribution to anti-cancer activity, many reports point at its ambivalent nature. CD40L enhance release of strongly pro-angiogenic factor, vascular endothelial growth factor (VEGF), and activator of coagulation, TF, the level of which is correlated with tumor metastasis. CD40L involvement in the inhibition of tumor progression has led to the emergence of not only therapy using recombinant forms of the ligand and vaccines in the treatment of cancer but also therapy consisting of inhibiting platelets-main source of CD40L. This article is a review of studies on the ambivalent role of CD40L in neoplastic diseases. PMID:25117071

  2. Mechanisms of Nifedipine-Downregulated CD40L/sCD40L Signaling in Collagen Stimulated Human Platelets.

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    Tso-Hsiao Chen

    Full Text Available The platelet-derived soluble CD40L (sCD40L release plays a critical role in the development of atherosclerosis. Nifedipine, a dihydropyridine-based L-type calcium channel blocker (CCB, has been reported to have an anti-atherosclerotic effect beyond its blood pressure-lowering effect, but the molecular mechanisms remain unclear. The present study was designed to investigate whether nifedipine affects sCD40L release from collagen-stimulated human platelets and to determine the potential role of peroxisome proliferator-activated receptor-β/-γ (PPAR-β/-γ. We found that treatment with nifedipine significantly inhibited the platelet surface CD40L expression and sCD40L release in response to collagen, while the inhibition was markedly reversed by blocking PPAR-β/-γ activity with specific antagonist such as GSK0660 and GW9662. Meanwhile, nifedipine also enhanced nitric oxide (NO and cyclic GMP formation in a PPAR-β/-γ-dependent manner. When the NO/cyclic GMP pathway was suppressed, nifedipine-mediated inhibition of sCD40L release was abolished significantly. Collagen-induced phosphorylation of p38MAPK, ERK1/2 and HSP27, matrix metalloproteinase-2 (MMP-2 expression/activity and reactive oxygen species (ROS formation were significantly inhibited by nifedipine, whereas these alterations were all attenuated by co-treatment with PPAR-β/-γ antagonists. Collectively, these results demonstrate that PPAR-β/-γ-dependent pathways contribute to nifedipine-mediated downregulation of CD40L/sCD40L signaling in activated platelets through regulation of NO/ p38MAPK/ERK1/2/HSP27/MMP-2 signalings and provide a novel mechanism regarding the anti-atherosclerotic effect of nifedipine.

  3. Targeted gene editing restores regulated CD40L function in X-linked hyper-IgM syndrome.

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    Hubbard, Nicholas; Hagin, David; Sommer, Karen; Song, Yumei; Khan, Iram; Clough, Courtnee; Ochs, Hans D; Rawlings, David J; Scharenberg, Andrew M; Torgerson, Troy R

    2016-05-26

    Loss of CD40 ligand (CD40L) expression or function results in X-linked hyper-immunoglobulin (Ig)M syndrome (X-HIGM), characterized by recurrent infections due to impaired immunoglobulin class-switching and somatic hypermutation. Previous attempts using retroviral gene transfer to correct murine CD40L expression restored immune function; however, treated mice developed lymphoproliferative disease, likely due to viral-promoter-dependent constitutive CD40L expression. These observations highlight the importance of preserving endogenous gene regulation in order to safely correct this disorder. Here, we report efficient, on-target, homology-directed repair (HDR) editing of the CD40LG locus in primary human T cells using a combination of a transcription activator-like effector nuclease-induced double-strand break and a donor template delivered by recombinant adeno-associated virus. HDR-mediated insertion of a coding sequence (green fluorescent protein or CD40L) upstream of the translation start site within exon 1 allowed transgene expression to be regulated by endogenous CD40LG promoter/enhancer elements. Additionally, inclusion of the CD40LG 3'-untranslated region in the transgene preserved posttranscriptional regulation. Expression kinetics of the transgene paralleled that of endogenous CD40L in unedited T cells, both at rest and in response to T-cell stimulation. The use of this method to edit X-HIGM patient T cells restored normal expression of CD40L and CD40-murine IgG Fc fusion protein (CD40-muIg) binding, and rescued IgG class switching of naive B cells in vitro. These results demonstrate the feasibility of engineered nuclease-directed gene repair to restore endogenously regulated CD40L, and the potential for its use in T-cell therapy for X-HIGM syndrome. PMID:26903548

  4. NORE1A induction by membrane-bound CD40L (mCD40L) contributes to CD40L-induced cell death and G1 growth arrest in p21-mediated mechanism.

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    Elmetwali, T; Salman, A; Palmer, D H

    2016-01-01

    Membrane-bound CD40L (mCD40L) but not soluble CD40L (sCD40L) has been implicated in direct cell death induction and apoptosis in CD40-expressing carcinomas. In this study, we show that mCD40L but not sCD40L induces NORE1A/Rassf5 expression in an NFκB-dependant mechanism. NORE1A expression appeared to contribute to mCD40L-induced cell death and enhance cell transition from G1 to S phase of the cell cycle in a p21-dependent mechanism. The upregulation of p21 protein was attributed to NORE1A expression, since NORE1A inhibition resulted in p21 downregulation. p21 upregulation was concomitant with lower p53 expression in the cytoplasmic fraction with no detectable increase at the nuclear p53 level. Moreover, mCD40L-induced cell death mediated by NORE1A expression appeared to be independent of mCD40L-induced cell death mediated by sustained JNK activation since NORE1A inhibition did not affect JNK phosphorylation and vice versa. The presented data allow better understanding of the mechanism by which mCD40L induces cell death which could be exploited in the clinical development of CD40-targeted anti-cancer therapies. PMID:26986513

  5. Multifunctional CD40L: pro- and anti-neoplastic activity

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    Korniluk, Aleksandra; Kemona, Halina; Dymicka-Piekarska, Violetta

    2014-01-01

    The CD40 ligand is a type I transmembrane protein that belongs to a tumor necrosis factor (TNF) superfamily. It is present not only on the surface of activated CD4+ T cells, B cells, blood platelets, monocytes, and natural killer (NK) cells but also on cancer cells. The receptor for ligand is constitutively expressed on cells, TNF family protein: CD40. The role of the CD40/CD40L pathway in the induction of body immunity, in inflammation, or in hemostasis has been well documented, whereas its ...

  6. Stratification of ST-elevation myocardial infarction patients based on soluble CD40L longitudinal changes.

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    Napoleão, Patrícia; Cabral, Luís B P; Selas, Mafalda; Freixo, Cláudia; Monteiro, Maria do Céu; Criado, Maria Begoña; Costa, Marina C; Enguita, Francisco J; Viegas-Crespo, Ana Maria; Saldanha, Carlota; Carmo, Miguel Mota; Ferreira, Rui Cruz; Pinheiro, Teresa

    2016-10-01

    Involvement of soluble CD40 ligand (sCD40L) in thrombosis and inflammation on the context of coronary artery disease is currently being revised. In that perspective, we had studied the association of sCD40L with markers of platelet activation and markers of endothelial and vascular function. On that cohort, a stratification of patients with acute myocardial infarction (AMI) 1 month after percutaneous coronary intervention (PCI) was observed based on concentrations of sCD40L. The study intended to identify the groups of AMI patients with different profiles of sCD40L concentrations and verify how medication, clinical evolution, biochemical data, and markers of regulation of endothelial function at genetic (endothelial nitric oxide synthase polymorphisms) and post-transcriptional levels (circulating microRNAs) affect sCD40L serum levels. Lower quartiles of sCD40L (polymorphism, and altered expression of a set of microRNAs assumed to be involved in the regulation of endothelial and cardiac function and myocardium hypertrophy, relative to patients in sCD40L upper quartiles. A characteristic sCD40L variation pattern in STEMI patients was identified. Low levels of sCD40L 1 month after PCI distinguish STEMI patients with worse prognosis, a compromised cardiac healing, and a persistent endothelial dysfunction, as given by the association between sCD40L, NT-proBNP, G894T polymorphism, and specific profile of miRNA expression. These results suggest sCD40L could have a prognostic value in STEMI patients. PMID:27172386

  7. Is Delayed Pressure Urticaria Associated with Increased Systemic Release of sCD40L?

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    T. Jasinska

    2013-01-01

    Full Text Available Background. Elevated levels of soluble CD40 Ligand (sCD40L were found in serum but not in plasma of patients with chronic spontaneous urticaria (CU. What is important is that sCD40L has proinflammatory properties, and its elevated plasma level may indicate increased risk of cardiovascular events. These observations should stimulate further evaluation of sCD40L in different forms of urticaria. Aim. In the present study, sCD40L plasma level was investigated in delayed pressure urticaria (DPU. Methods. As platelets are predominant and variable sources of sCD40L, we investigated sCD40L concentration in platelet-poor plasma (PPP, which seems the best way to minimize the potential contribution of these cells to the ligand level. Results. Plasma sCD40L concentration was significantly increased in the DPU group compared to the healthy controls. Conclusions. It seems that DPU is associated with increased systemic release of sCD40L, which is believed to derive predominantly from activated platelets. The present study as well as the earlier contributions suggest that distinct cells activity, including platelets, may be identified in different types of urticaria.

  8. Role for CD40L in the Therapy of Human Cancer

    Institute of Scientific and Technical Information of China (English)

    Feng Wei; Xiubao Ren; Xishan Hao

    2005-01-01

    CD40L-CD40 interaction is central to the control of thymusdependent humoral .immunity and cell mediated immune responses.CD40, a member of the tumor necrosis factor receptor (TNF- R) family,has been found on the surface of B lymphocytes, monocytes, hematopoietic progenitors, dendritic cells (DCs), endothelial cells, epithelial cells and so on. Its natural ligand (CD40 ligand, CD40L), CD154, a member of the tumor necrosis factor (TNF) family, is mainly expressed on activated CD4+ T lymphocytes. A direct growth-inhibitory effect can be found when ligated CD40 is on human breast, ovarian, cervical, bladder, non-small cell lung, and squamous epithelial carcinoma cells. This effect is related to the induction of cell cycle blockage and/or apoptosis. CD40L induces phenotypic and functional maturation of DCs, and can increase tumor immunogenicity through up-regulation of costimulatory molecular expression and cytokine production by epithelial cancer cells. As a result,CD40L can enhance tumor rejection immune responses. Furthermore, by means of a "bystander effect", even CD40-negative tumor subsets can be eliminated by activated tumor-reactive cytotoxic T lymphocytes(CTL),This review summarizes recent findings on CD40L recombinant protein and gene therapy-based tumor treatment approaches.

  9. Evaluation of CD40, its ligand CD40L and Bcl-2 in psoriatic patients

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    Bożena Chodynicka

    2012-04-01

    Full Text Available Psoriasis is a chronic, recurrent, inflammatory disease. Recent investigations indicate an autoimmune pathogenesis of the disease. Apoptosis plays an important role in the regulation of immune mechanisms in many autoimmune diseases. Although CD40, CD40L, and Bcl-2 have already been studied in psoriatic skin lesions, little is known about their circulating forms. The aim of the present study was to evaluate the serum concentrations of Bcl-2, soluble CD40 and CD40L in psoriatic patients. The study was performed using ELISA kits in 39 psoriatic patients before treatment and after two weeks of topical ointment. Data was analyzed with respect to severity of psoriasis, duration of the disease, and coexisting psoriatic arthritis. Our results revealed that serum concentrations of soluble CD40 and CD40L before and after treatment were significantly higher (p < 0.01 and p < 0.001 in patients with psoriasis compared to the control group. Topical treatment of psoriatic lesions with dithranol ointment failed to decrease serum of CD40 and CD40L, which has not been described until now. There was no significant difference in serum Bcl-2 concentration between the compared groups. We did not find significant differences in serum concentrations of Bcl-2, CD40 or CD40L between patients with mild or severe psoriasis, nor any correlation between disease duration and the presence of psoriatic arthritis symptoms. Our data indicates upregulation of the CD40/CD40L system in psoriatic patients despite topical treatment and suggests their possible role in the pathogenesis of psoriasis.

  10. Soluble CD40L in children and adolescents with type 1 diabetes: relation to microvascular complications and glycemic control.

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    El-Asrar, Mohamed A; Adly, Amira Am; Ismail, Eman A

    2012-12-01

    CD40-soluble CD40 ligand (sCD40L) interactions might constitute an important mediator for vascular inflammation that initiates diabetic microangiopathy. Little is known about the relation between sCD40L and glycemic control. Therefore, this study aimed to evaluate sCD40L levels in patients with type 1 diabetes and its relation to microvascular complications and metabolic control. Sixty patients with type 1 diabetes were compared with 30 healthy control subjects. Detailed medical history, thorough clinical examination, and laboratory assessment of high-sensitivity C-reactive protein, glycemic control, and the presence of microvascular complications were performed. Measurement of serum sCD40L levels was done using enzyme-linked immunosorbent assay. Patients were divided into two groups according to the presence of microvascular complications. Serum sCD40L levels were significantly elevated in patients with type 1 diabetes in both groups compared with healthy controls (p < 0.001). Patients with microvascular complications had higher serum sCD40L concentrations than non-complicated cases (median, 13 000 vs. 450 pg/mL; p < 0.001). Serum sCD40L cutoff value of 530 pg/mL was able to differentiate complicated from non-complicated cases (p < 0.001). Patients with microalbuminuria or peripheral neuropathy showed higher levels of sCD40L when compared with patients without these complications (p < 0.05). Serum sCD40L levels were positively correlated with hemoglobin A1c and urinary albumin excretion (p < 0.001). We suggest that serum sCD40L levels are elevated in type 1 diabetes, particularly in patients with microvascular complications and a significant correlation with glycemic control exists. Therefore, measurement of serum sCD40L levels in poorly controlled patients would help to identify those at high risk of developing microvascular complications.

  11. CD40L Deficiency Ameliorates Adipose Tissue Inflammation and Metabolic Manifestations of Obesity in Mice

    NARCIS (Netherlands)

    M. Poggi; D. Engel; A. Christ; L. Beckers; E. Wijnands; L. Boon; A. Driessen; J. Cleutjens; C. Weber; N. Gerdes; E. Lutgens

    2011-01-01

    Obese adipose tissue shows hallmarks of chronic inflammation, which promotes the development of metabolic disorders. The mechanisms by which immune cells interact with each other or with metabolism-associated cell types, and the players involved, are still unclear. The CD40-CD40L costimulatory dyad

  12. Enhanced Soluble Serum CD40L and Serum P-Selectin Levels in Primary Aldosteronism.

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    Petramala, L; Iacobellis, G; Carnevale, R; Marinelli, C; Zinnamosca, L; Concistrè, A; Galassi, M; Iannucci, G; Lucia, P; Pignatelli, P; Ciardi, A; Violi, F; De Toma, G; Letizia, C

    2016-07-01

    Primary aldosteronism (PA) is one of the most frequent forms of secondary hypertension, associated with atherosclerosis and higher risk of cardiovascular events. Platelets play a key role in the atherosclerotic process. The aim of the study was to evaluate the platelet activation by measuring serum levels of soluble CD40L (sCD40L) and P-selectin (sP-selectin) in consecutive PA patients [subgroup: aldosterone-secreting adrenal adenoma (APA) and bilateral adrenal hyperplasia (IHA)], matched with essential hypertensive (EH) patients. The subgroup of APA patients was revaluated 6-months after unilateral adrenalectomy. In all PA group, we measured higher serum levels of both sP-selectin (14.29±9.33 pg/ml) and sCD40L (9.53±4.2 ng/ml) compared to EH patients (9.39±5.3 pg/ml and 3.54±0.94 ng/ml, respectively; pAPA, PA patients showed significant reduction of blood pressure (BP) values, plasma aldosterone (PAC) levels and ARR-ratio, associated with a significant reduction of sP-selectin (16.74±8.9 pg/ml vs. 8.1±3.8 pg/ml; pAPA patients (r=0.54; pAPA patients. PMID:27101095

  13. Immune response is an important aspect of the antitumor effect produced by a CD40L-encoding oncolytic adenovirus.

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    Diaconu, Iulia; Cerullo, Vincenzo; Hirvinen, Mari L M; Escutenaire, Sophie; Ugolini, Matteo; Pesonen, Saila K; Bramante, Simona; Parviainen, Suvi; Kanerva, Anna; Loskog, Angelica S I; Eliopoulos, Aristides G; Pesonen, Sari; Hemminki, Akseli

    2012-05-01

    Oncolytic adenovirus is an attractive platform for immunotherapy because virus replication is highly immunogenic and not subject to tolerance. Although oncolysis releases tumor epitopes and provides costimulatory danger signals, arming the virus with immunostimulatory molecules can further improve efficacy. CD40 ligand (CD40L, CD154) induces apoptosis of tumor cells and triggers several immune mechanisms, including a T-helper type 1 (T(H)1) response, which leads to activation of cytotoxic T cells and reduction of immunosuppression. In this study, we constructed a novel oncolytic adenovirus, Ad5/3-hTERT-E1A-hCD40L, which features a chimeric Ad5/3 capsid for enhanced tumor transduction, a human telomerase reverse transcriptase (hTERT) promoter for tumor selectivity, and human CD40L for increased efficacy. Ad5/3-hTERT-E1A-hCD40L significantly inhibited tumor growth in vivo via oncolytic and apoptotic effects, and (Ad5/3-hTERT-E1A-hCD40L)-mediated oncolysis resulted in enhanced calreticulin exposure and HMGB1 and ATP release, which were suggestive of immunogenicity. In two syngeneic mouse models, murine CD40L induced recruitment and activation of antigen-presenting cells, leading to increased interleukin-12 production in splenocytes. This effect was associated with induction of the T(H)1 cytokines IFN-γ, RANTES, and TNF-α. Tumors treated with Ad5/3-CMV-mCD40L also displayed an enhanced presence of macrophages and cytotoxic CD8(+) T cells but not B cells. Together, our findings show that adenoviruses coding for CD40L mediate multiple antitumor effects including oncolysis, apoptosis, induction of T-cell responses, and upregulation of T(H)1 cytokines.

  14. Expression of CD40 and CD40L in Gastric Cancer Tissue and Its Clinical Significance

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    Rui Li

    2009-09-01

    Full Text Available To study expression of CD40 and CD40L in gastric cancer tissue we assessed gastric cancer patients admitted to the Department of Gastroenterology of The First Affiliated Hospital of Soochow University and control subjects. Gastric cancer and normal (from around tumours tissue samples were obtained from patients. Venous blood samples (gastric cancer and ulcer groups were drawn on the morning of the day before surgery for the measurement of peripheral sCD40L. The expression of CD40 in gastric carcinoma specimens was examined immuno-histochemically. The clinicopathological factors, including age, sex, tumor size, gross appearance, degree of cellular differentiation, histological classification, depth of tumor invasion, lymph node metastasis, peritoneal dissemination, and TNM stage were analyzed according to the different expression of CD40. The results indicated a high CD40 expression in gastric cancer tissues. This positive expression of CD40 revealed a significant (P < 0.05 correlation with lymphatic metastasis and tumor TNM stage in gastric cancer patients. It is concluded that higher CD40 expression existed in expanding type tumors and could play an important role in clinical diagnosis of gastric cancer patients.

  15. Excess soluble CD40L contributes to blood brain barrier permeability in vivo: implications for HIV-associated neurocognitive disorders.

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    Donna C Davidson

    Full Text Available Despite the use of anti-retroviral therapies, a majority of HIV-infected individuals still develop HIV-Associated Neurocognitive Disorders (HAND, indicating that host inflammatory mediators, in addition to viral proteins, may be contributing to these disorders. Consistently, we have previously shown that levels of the inflammatory mediator soluble CD40L (sCD40L are elevated in the circulation of HIV-infected, cognitively impaired individuals as compared to their infected, non-impaired counterparts. Recent studies from our group suggest a role for the CD40/CD40L dyad in blood brain barrier (BBB permeability and interestingly, sCD40L is thought to regulate BBB permeability in other inflammatory disorders of the CNS. Using complementary multiphoton microscopy and quantitative analyses in wild-type and CD40L deficient mice, we now reveal that the HIV transactivator of transcription (Tat can induce BBB permeability in a CD40L-dependent manner. This permeability of the BBB was found to be the result of aberrant platelet activation induced by Tat, since depletion of platelets prior to treatment reversed Tat-induced BBB permeability. Furthermore, Tat treatment led to an increase in granulocyte antigen 1 (Gr1 positive monocytes, indicating an expansion of the inflammatory subset of cells in these mice, which were found to adhere more readily to the brain microvasculature in Tat treated animals. Exploring the mechanisms by which the BBB becomes compromised during HIV infection has the potential to reveal novel therapeutic targets, thereby aiding in the development of adjunct therapies for the management of HAND, which are currently lacking.

  16. Excess soluble CD40L contributes to blood brain barrier permeability in vivo: implications for HIV-associated neurocognitive disorders.

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    Davidson, Donna C; Hirschman, Michael P; Sun, Anita; Singh, Meera V; Kasischke, Karl; Maggirwar, Sanjay B

    2012-01-01

    Despite the use of anti-retroviral therapies, a majority of HIV-infected individuals still develop HIV-Associated Neurocognitive Disorders (HAND), indicating that host inflammatory mediators, in addition to viral proteins, may be contributing to these disorders. Consistently, we have previously shown that levels of the inflammatory mediator soluble CD40L (sCD40L) are elevated in the circulation of HIV-infected, cognitively impaired individuals as compared to their infected, non-impaired counterparts. Recent studies from our group suggest a role for the CD40/CD40L dyad in blood brain barrier (BBB) permeability and interestingly, sCD40L is thought to regulate BBB permeability in other inflammatory disorders of the CNS. Using complementary multiphoton microscopy and quantitative analyses in wild-type and CD40L deficient mice, we now reveal that the HIV transactivator of transcription (Tat) can induce BBB permeability in a CD40L-dependent manner. This permeability of the BBB was found to be the result of aberrant platelet activation induced by Tat, since depletion of platelets prior to treatment reversed Tat-induced BBB permeability. Furthermore, Tat treatment led to an increase in granulocyte antigen 1 (Gr1) positive monocytes, indicating an expansion of the inflammatory subset of cells in these mice, which were found to adhere more readily to the brain microvasculature in Tat treated animals. Exploring the mechanisms by which the BBB becomes compromised during HIV infection has the potential to reveal novel therapeutic targets, thereby aiding in the development of adjunct therapies for the management of HAND, which are currently lacking.

  17. CD40L induces multidrug resistance to apoptosis in breast carcinoma and lymphoma cells through caspase independent and dependent pathways

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    Blay Jean-Yves

    2006-03-01

    Full Text Available Abstract Background CD40L was found to reduce doxorubicin-induced apoptosis in non Hodgkin's lymphoma cell lines through caspase-3 dependent mechanism. Whether this represents a general mechanism for other tumor types is unknown. Methods The resistance induced by CD40L against apoptosis induced by a panel of cytotoxic chemotherapeutic drugs in non Hodgkin's lymphoma and breast carcinoma cell lines was investigated. Results Doxorubicin, cisplatyl, etoposide, vinblastin and paclitaxel increased apoptosis in a dose-dependent manner in breast carcinoma as well as in non Hodgkin's lymphoma cell lines. Co-culture with irradiated L cells expressing CD40L significantly reduced the percentage of apoptotic cells in breast carcinoma and non Hodgkin's lymphoma cell lines treated with these drugs. In breast carcinoma cell lines, these 5 drugs induced an inconsistent increase of caspase-3/7 activity, while in non Hodgkin's lymphoma cell lines all 5 drugs increased caspase-3/7 activity up to 28-fold above baseline. Co-culture with CD40L L cells reduced (-39% to -89% the activation of caspase-3/7 induced by these agents in all 5 non Hodgkin's lymphoma cell lines, but in none of the 2 breast carcinoma cell lines. Co culture with CD40L L cells also blocked the apoptosis induced by exogenous ceramides in breast carcinoma and non Hodgkin's lymphoma cell lines through a caspase-3-like, 8-like and 9-like dependent pathways. Conclusion These results indicate that CD40L expressed on adjacent non tumoral cells induces multidrug resistance to cytotoxic agents and ceramides in both breast carcinoma and non Hodgkin's lymphoma cell lines, albeit through a caspase independent and dependent pathway respectively.

  18. CD40L deficiency attenuates diet-induced adipose tissue inflammation by impairing immune cell accumulation and production of pathogenic IgG-antibodies.

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    Dennis Wolf

    Full Text Available BACKGROUND: Adipose tissue inflammation fuels the metabolic syndrome. We recently reported that CD40L--an established marker and mediator of cardiovascular disease--induces inflammatory cytokine production in adipose cells in vitro. Here, we tested the hypothesis that CD40L deficiency modulates adipose tissue inflammation in vivo. METHODOLOGY/PRINCIPAL FINDINGS: WT or CD40L(-/- mice consumed a high fat diet (HFD for 20 weeks. Inflammatory cell recruitment was impaired in mice lacking CD40L as shown by a decrease of adipose tissue macrophages, B-cells, and an increase in protective T-regulatory cells. Mechanistically, CD40L-deficient mice expressed significantly lower levels of the pro-inflammatory chemokine MCP-1 both, locally in adipose tissue and systemically in plasma. Moreover, levels of pro-inflammatory IgG-antibodies against oxidized lipids were reduced in CD40L(-/- mice. Also, circulating low-density lipoproteins and insulin levels were lower in CD40L(-/- mice. However, CD40L(-/- mice consuming HFD were not protected from the onset of diet-induced obesity (DIO, insulin resistance, and hepatic steatosis, suggesting that CD40L selectively limits the inflammatory features of diet-induced obesity rather than its metabolic phenotype. Interestingly, CD40L(-/- mice consuming a low fat diet (LFD showed both, a favorable inflammatory and metabolic phenotype characterized by diminished weight gain, improved insulin tolerance, and attenuated plasma adipokine levels. CONCLUSION: We present the novel finding that CD40L deficiency limits adipose tissue inflammation in vivo. These findings identify CD40L as a potential mediator at the interface of cardiovascular and metabolic disease.

  19. PU.1 Expression in T Follicular Helper Cells Limits CD40L-Dependent Germinal Center B Cell Development.

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    Awe, Olufolakemi; Hufford, Matthew M; Wu, Hao; Pham, Duy; Chang, Hua-Chen; Jabeen, Rukhsana; Dent, Alexander L; Kaplan, Mark H

    2015-10-15

    PU.1 is an ETS family transcription factor that is important for the development of multiple hematopoietic cell lineages. Previous work demonstrated a critical role for PU.1 in promoting Th9 development and in limiting Th2 cytokine production. Whether PU.1 has functions in other Th lineages is not clear. In this study, we examined the effects of ectopic expression of PU.1 in CD4(+) T cells and observed decreased expression of genes involved with the function of T follicular helper (Tfh) cells, including Il21 and Tnfsf5 (encoding CD40L). T cells from conditional mutant mice that lack expression of PU.1 in T cells (Sfpi1(lck-/-)) demonstrated increased production of CD40L and IL-21 in vitro. Following adjuvant-dependent or adjuvant-independent immunization, we observed that Sfpi1(lck-/-) mice had increased numbers of Tfh cells, increased germinal center B cells (GCB cells), and increased Ab production in vivo. This correlated with increased expression of IL-21 and CD40L in Tfh cells from Sfpi1(lck-/-) mice compared with control mice. Finally, although blockade of IL-21 did not affect GCB cells in Sfpi1(lck-/-) mice, anti-CD40L treatment of immunized Sfpi1(lck-/-) mice decreased GCB cell numbers and Ag-specific Ig concentrations. Together, these data indicate an inhibitory role for PU.1 in the function of Tfh cells, germinal centers, and Tfh-dependent humoral immunity.

  20. Increased concentrations of soluble vascular cell adhesion molecule-1 and soluble CD40L in subjects with metabolic syndrome.

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    Palomo, Iván G; Jaramillo, Julio C; Alarcón, Marcelo L; Gutiérrez, César L; Moore-Carrasco, Rodrigo; Segovia, Fabián M; Leiva, Elba M; Mujica, Verónica E; Icaza, Gloria; Dí, Nora S

    2009-01-01

    Metabolic syndrome (MS) is associated with a high incidence rate of cardiovascular disease. It is characterized by abdominal obesity, elevated blood pressure, atherogenic dyslipidemia [high LDL-c (low density lipoprotein cholesterol) and low HDL-c (high density lipoprotein cholesterol)] and insulin resistance or glucose intolerance. In the context of MS, alterations in the plasmatic levels of some soluble forms of cell adhesion molecules can appear, e.g., soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble E-selectin (sE-selectin) and soluble CD40L (sCD40L). The objective of this study was to compare the serum levels of sVCAM-1, sE-selectin and sCD40L in MS and non-MS groups and to associate these molecules with the diagnostic criteria of MS. A total of 185 non-smokers between 45 and 64 years of age were included. Of these, 93 corresponded to the MS group and the remaining 92 to a non-MS group (according to modified ATP III criteria). The serum concentration of sVCAM-1, sE-selectin and sCD40L was determined by commercial solid phase ELISA. The results were expressed as a median and interquartile range. The MS group showed high levels of sVCAM-1 (558.9 ng/ml; 481.3-667.6 ng/ml) compared with the non-MS group (405.2 ng/ml; 361.0-470.5 ng/ml) (p<0.0001). As well, the median level of sCD40L (3.0 ng/ml; 2.1l-11.7 ng/ml) was significantly higher in the MS group than that in the non-MS group (2.6 ng/ml; 2.3-3.4 ng/ml) (p=0.0061). sE-selectin levels did not differ significantly between the groups: 73.9 ng/ml (58.3-87.0 ng/ml) and 68.5 ng/ml (51.6-97.5 ng/ml) in the MS and non-MS group, respectively. In conclusion, the serum levels of sVCAM-1 and sCD40L, but not sE-selectin, were significantly higher in patients with MS than in subjects that did not present MS. MS may therefore increase the expression of cell adhesion molecules, probably through endothelial activation. PMID:21475854

  1. Regulatory T Cell-Dependent and -Independent Mechanisms of Immune Suppression by CD28/B7 and CD40/CD40L Costimulation Blockade.

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    Vogel, Isabel; Verbinnen, Bert; Van Gool, Stefaan; Ceuppens, Jan L

    2016-07-15

    Blocking of costimulatory CD28/B7 and CD40/CD40L interactions is an experimental approach to immune suppression and tolerance induction. We previously reported that administration of a combination of CTLA-4Ig and MR1 (anti-CD40L mAb) for blockade of these interactions induces tolerance in a fully mismatched allogeneic splenocyte transfer model in mice. We now used this model to study whether regulatory T cells (Tregs) contribute to immune suppression and why both pathways have to be blocked simultaneously. Mice were injected with allogeneic splenocytes, CD4(+) T cells, or CD8(+) T cells and treated with MR1 mAb and different doses of CTLA-4Ig. The graft-versus-host reaction of CD4(+) T cells, but not of CD8(+) T cells, was inhibited by MR1. CTLA-4Ig was needed to cover CD8(+) T cells but had only a weak effect on CD4(+) T cells. Consequently, only the combination provided full protection when splenocytes were transferred. Importantly, MR1 and low-dose CTLA-4Ig treatment resulted in a relative increase in Tregs, and immune suppressive efficacy was abolished in the absence of Tregs. High-dose CTLA-4Ig treatment, in contrast, prevented Treg expansion and activity, and in combination with MR1 completely inhibited CD4(+) and CD8(+) T cell activation in a Treg-independent manner. In conclusion, MR1 and CTLA-4Ig act synergistically as they target different T cell populations. The contribution of Tregs to immune suppression by costimulation blockade depends on the concentration of CTLA-4Ig and thus on the degree of available CD28 costimulation. PMID:27288533

  2. CD40/CD40L Dyad in the Inflammatory and Immune Responses in the Central Nervous System

    Institute of Scientific and Technical Information of China (English)

    Keqiang Chen; Jian Huang; Wanghua Gong; Lingzhi Zhang; Peichu Yu; Ji Ming Wang

    2006-01-01

    CD40 and its cognate ligand (CD40L) are a pair of regulators of pro-inflammatory and immune responses. In the central nervous system (CNS), CD40 is expressed on a variety of cells, including vascular endothelial cells, smooth muscle cells, astrocytes and microglia (the brain macrophages, being the most sensitive cell type to respond to CD40 ligand). Interaction between CD40 on microglia and CD40L presented by infiltrating T lymphocytes and other resident CNS cells triggers a series of intracellular signaling events that promote the production of a wide array of cytokines, chemokines and neurotoxins. Thus, both molecules serve as amplifiers of pro-inflammatory and immune responses in the CNS and constitute important molecular targets for therapeutic intervention of diseases.

  3. High sCD40L levels Early After Trauma are Associated with Enhanced Shock, Sympathoadrenal Activation, Tissue and Endothelial Damage, Coagulopathy and Mortality

    DEFF Research Database (Denmark)

    Johansson, P I; Sørensen, A M; Perner, A;

    2012-01-01

    Background: Severe injury activates the sympathoadrenal, hemostatic and inflammatory systems, but a maladapted response may contribute to poor outcome. Soluble CD40L is a platelet derived mediator that links inflammation, hemostasis and vascular dysfunction. Objectives: To investigate the associa......Background: Severe injury activates the sympathoadrenal, hemostatic and inflammatory systems, but a maladapted response may contribute to poor outcome. Soluble CD40L is a platelet derived mediator that links inflammation, hemostasis and vascular dysfunction. Objectives: To investigate...

  4. Assessment of serum levels of soluble CD40L in Egyptian children and adolescents with type 1 diabetes mellitus: Relationship to microalbuminuria and glycemic control

    Directory of Open Access Journals (Sweden)

    Kotb Abbass Metwalley

    2013-01-01

    Full Text Available Context: Soluble CD40 ligand (sCD40L is known to be elevated in different clinical situations including hypercholesterolemia, acute coronary syndromes, and type 2 diabetes mellitus (T2DM, Data about the relationship between type 1 diabetes mellitus (T1DM and sCD40L is limited. In addition, the potential role ofsCD40Lin the pathogenesis of vascular complications in children and adolescents with T1DM is to be clarified. Hence, the study aimed at assessment of sCD40L levels in children and adolescents with T1DM and correlation of these levels with glycemic control and microalbuminuria. Settings and Design: Cross-sectional controlled study. Materials and Methods: The study was performed in the Pediatric Endocrinology and Diabetes Unit, Assuit University Children Hospital, Assiut, Egypt. It included 70 children and adolescents with T1DM (mean age 14. 76 ± 2.21 years. Cases were further subdivided into 43 cases with normoalbuminuria and 27 cases with microalbuminuria according to presence or absence or microalbuminuria in fresh urine samples. Twentyfive healthy subjects, age- and sex-matched were included as control group (mean age = 13.62 ± 2.11 years. Studied cases were subjected to medical history, clinical examination, and laboratory assessment of fasting blood glucose (FBG, lipid profile, glycosylated hemoglobin (HbA1c, and sCD40L were performed. Results: Mean HbA1c and sCD40L were significantly higher in diabetic children (n = 70 compared to control (n = 25 (P < 0.001 for each. Mean HbA1c and sCD40L levels were significantly higher in microalbuminuric cases (n = 27 compared to normoalbuminuric cases (n = 43 (P < 0.05 and <0.01, respectively.We also observed a significant positive correlation between sCD40L levels and the age, diabetes duration, HbA1c, and urinary albumin creatinine ratio. Conclusions: The high serum sCD40L levels in children and adolescents with T1DM particularly in those with microalbminuria and its positive correlation with

  5. Rola szlaku CD40/CD40L w biologicznej aktywności płytek krwi. Część II

    Directory of Open Access Journals (Sweden)

    Joanna Saluk-Juszczak

    2010-12-01

    Full Text Available The main function of blood platelets is their haemostatic role, but recent evidence shows that processes ofplatelet activation may be sometimes a critical link between haemostasis and development of inflammation.The discovery of expression of CD40 and its ligand – CD40L on platelet surface revealed the participation ofthese cells in immune responses and inflammation. In many pathological processes related to cardiovasculardisorders, e.g. changes connected with menopause, enhanced platelet activation is observed in atherosclerosis,diabetes and cancer, in which proinflammatory and prothrombotic CD40/CD40L pathway is involved. Signalsthat drive inflammatory events and are transmitted through CD40/CD40L pathway cause the inflammatory cellinteractions leading to induction of innate immune response. This signal transduction is associated with therelease of adhesion molecules, chemokines, cytokines, tissue factors, reactive oxygen species, growth factorsand other proinflammatory mediators from activated cells. Hyperactivity of platelets associated with signaltransduction by CD40/CD40L pathway is observed in many pathogenic processes, including thrombosis, diabetes,inflammation and cancers. The inhibition of CD40/CD40L pathway may provide alternative treatment forvarious diseases in the future.

  6. Reduced CD40L expression on ex vivo activated CD4+T-lymphocytes from patients with excellent renal allograft function measured with a rapid whole blood flow cytometry procedure

    OpenAIRE

    Lederer, Stephan R.; Friedrich, N; Gruber, R; Landgraf, R; Toepfer, Marcel; Sitter, Thomas

    2004-01-01

    Background: The CD40-CD40L (CD154) costimulatory pathway plays a critical role in the pathogenesis of kidney allograft rejection. In renal transplant biopsies, CD4+ CD40L+ graft-infiltrating cells were detected during chronic rejection in contrast to acute rejection episodes. Using a rapid noninvasive FACS procedure, we were able to demonstrate CD40L upregulation in peripheral blood of patients with chronic renal allograft dysfunction. Materials and Methods: Whole blood from recipients of ren...

  7. Valproic acid inhibits the release of soluble CD40L induced by non-nucleoside reverse transcriptase inhibitors in human immunodeficiency virus infected individuals.

    Directory of Open Access Journals (Sweden)

    Donna C Davidson

    Full Text Available Despite the use of highly active antiretroviral therapies (HAART, a majority of Human Immunodeficiency Virus Type 1 (HIV infected individuals continually develop HIV - Associated Neurocognitive Disorders (HAND, indicating that host inflammatory mediators, in addition to viral proteins, may be contributing to these disorders. Consistent with this notion, we have previously shown that levels of the inflammatory mediator soluble CD40 ligand (sCD40L are elevated in the plasma and cerebrospinal fluid (CSF of HIV infected, cognitively impaired individuals, and that excess sCD40L can contribute to blood brain barrier (BBB permeability in vivo, thereby signifying the importance of this inflammatory mediator in the pathogenesis of HAND. Here we demonstrate that the non-nucleoside reverse transcriptase inhibitor (NNRTI efavirenz (EFV induces the release of circulating sCD40L in both HIV infected individuals and in an in vitro suspension of washed human platelets, which are the main source of circulating sCD40L. Additionally, EFV was found to activate glycogen synthase kinase 3 beta (GSK3β in platelets, and we now show that valproic acid (VPA, a known GSK3β inhibitor, was able to attenuate the release of sCD40L in HIV infected individuals receiving EFV, and in isolated human platelets. Collectively these results have important implications in determining the pro-inflammatory role that some antiretroviral regimens may have. The use of antiretrovirals remains the best strategy to prevent HIV-associated illnesses, including HAND, however these drugs have clear limitations to this end, and thus, these results underscore the need to develop adjunctive therapies for HAND that can also minimize the undesired negative effects of the antiretrovirals.

  8. Agreement of skin test with IL-4 production and CD40L expression by T cells upon immunotherapy of subjects with systemic reactions to Hymenoptera stings.

    Science.gov (United States)

    Urra, José M; Cabrera, Carmen M; Alfaya, Teresa; Feo-Brito, Francisco

    2016-02-01

    Venom immunotherapy is the only curative intervention for subjects with Hymenoptera venom allergy who suffering systemic reactions upon bee or wasp stings. Venom immunotherapy can restore normal immunity against venom allergens, as well as providing to allergic subjects a lifetime tolerance against venoms. Nevertheless, it is necessary using safety assays to monitoring the development of tolerance in the VIT protocols to avoid fatal anaphylactic reactions. The purpose of this study was to assess the modifications in several markers of tolerance induction in subjects with Hymenoptera venom allergy undergoing immunotherapy. The studies were performed at baseline time and after six month of VIT. Intradermal skin tests, basophil activation tests, specific IgE levels; and the T-cell markers (IL-4 and IFN-γ producing cells; and expression of the surface activation markers CD40L and CTLA-4) were assayed. At six month of immunotherapy all parameters studied had significant alterations. All decreased, except the IFN-γ producing cells. In addition, modifications in intradermal skin test showed a significant correlation with both, CD40L expression on CD4 T lymphocytes (p=0.043) and IL-4 producing T lymphocytes (p=0.012). Neither basophil activation test nor serum levels of sIgE demonstrated any correlation with the immunological parameters studied nor among them. These results suggest that both IL-4 production and CD40L expression could be two good indicators of the beneficial effects of venom immunotherapy which translate into skin tests. PMID:26774053

  9. Maturation of dendritic cells by recombinant human CD40L-trimer leads to a homogeneous cell population with enhanced surface marker expression and increased cytokine production

    DEFF Research Database (Denmark)

    Würtzen, P A; Nissen, Mogens Holst; Claesson, M H

    2001-01-01

    -cell activating capacity of the DC. We studied DC phenotype and cytokine production as well as the T-cell proliferation and cytotoxic T lympocyte (CTL) activation induced by DC generated in vitro. In addition, the effect of exposure to recombinant human CD40L-trimer (huCD40LT) on these parameters was investigated....... Effective differentiation of monocytes derived from freshly isolated peripheral blood mononuclear cells (PBMC) was obtained with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4. The DC expression of human leucocyte antigen (HLA) molecules, CD80, CD83, and CD86 was markedly...

  10. T Cell Cancer Therapy Requires CD40-CD40L Activation of Tumor Necrosis Factor and Inducible Nitric-Oxide-Synthase-Producing Dendritic Cells.

    Science.gov (United States)

    Marigo, Ilaria; Zilio, Serena; Desantis, Giacomo; Mlecnik, Bernhard; Agnellini, Andrielly H R; Ugel, Stefano; Sasso, Maria Stella; Qualls, Joseph E; Kratochvill, Franz; Zanovello, Paola; Molon, Barbara; Ries, Carola H; Runza, Valeria; Hoves, Sabine; Bilocq, Amélie M; Bindea, Gabriela; Mazza, Emilia M C; Bicciato, Silvio; Galon, Jérôme; Murray, Peter J; Bronte, Vincenzo

    2016-09-12

    Effective cancer immunotherapy requires overcoming immunosuppressive tumor microenvironments. We found that local nitric oxide (NO) production by tumor-infiltrating myeloid cells is important for adoptively transferred CD8(+) cytotoxic T cells to destroy tumors. These myeloid cells are phenotypically similar to inducible nitric oxide synthase (NOS2)- and tumor necrosis factor (TNF)-producing dendritic cells (DC), or Tip-DCs. Depletion of immunosuppressive, colony stimulating factor 1 receptor (CSF-1R)-dependent arginase 1(+) myeloid cells enhanced NO-dependent tumor killing. Tumor elimination via NOS2 required the CD40-CD40L pathway. We also uncovered a strong correlation between survival of colorectal cancer patients and NOS2, CD40, and TNF expression in their tumors. Our results identify a network of pro-tumor factors that can be targeted to boost cancer immunotherapies.

  11. Direct Interaction of CD40 on Tumor Cells with CD40L on T Cells Increases the Proliferation of Tumor Cells by Enhancing TGF-β Production and Th17 Differentiation.

    Directory of Open Access Journals (Sweden)

    Hyemin Kim

    Full Text Available It has recently been reported that the CD40-CD40 ligand (CD40L interaction is important in Th17 development. In addition, transforming growth factor-beta (TGF-β promotes tumorigenesis as an immunosuppressive cytokine and is crucial in the development of Th17 cells. This study investigated the role of CD40 in breast cancer cells and its role in immunosuppressive function and tumor progression. CD40 was highly expressed in the breast cancer cell line MDA-MB231, and its stimulation with CD40 antibodies caused the up-regulation of TGF-β. Direct CD40-CD40L interaction between MDA-MB231 cells and activated T cells also increased TGF-β production and induced the production of IL-17, which accelerated the proliferation of MDA-MB231 cells through the activation of STAT3. Taken together, the direct CD40-CD40L interaction of breast tumor cells and activated T cells increases TGF-β production and the differentiation of Th17 cells, which promotes the proliferation of breast cancer cells.

  12. Estudio de la díada CD40/CD40L y su modulación por la adiponectina en el síndrome metabólico

    OpenAIRE

    Restituto-Aranguíbel, P. (Patricia); N. Varo

    2010-01-01

    En el contexto del concepto actual del Síndrome Metabólico como una enfermedad inflamatoria y protrombótica, el presente estudio muestra que la vía del CD40/CD40L puede desempeñar un papel en el mayor riesgo cardiovascular de estos pacientes. Hemos encontrado concentraciones elevadas de la forma soluble del ligando de CD40 en pacientes con Síndrome Metabólico, posiblemente procedente de las plaquetas. Estos niveles, se ha demostrado ampliamente en la bibliografía disponible que se asocian con...

  13. 咖啡对血小板糖蛋白ⅡbⅢa、CD40L表达的 急性影响%ACUTE EFFECT OF COFFEE ON EXPRESSION OF PLATELET GLYCOPROTEINS ⅡbⅢa,CD40L

    Institute of Scientific and Technical Information of China (English)

    刘磊; 潘娟娟; 凌怡; 潘力健; 龚辉

    2016-01-01

    Objective To investigate whether the consumption of coffee affects platelets function.Methods Twelve healthy men were enrolled in a 3-week crossover study. In 3 different sessions, subjects drank either coffee (containing 180 mg caffeine), or a caffeine tablet (180mg), or placebo. Platelet glycoproteins Ⅱb/Ⅲa (GPⅡb/Ⅲa), and CD40L expression were detected by flow cytometry at 0h, 2h, 4h. Effects of different concentrations of caffeine on platelet GPⅡb/Ⅲa were detected in vitro.Results Coffee drinking inhibited ADP induced GPⅡb/Ⅲa (P<0.05 VS control at 2h and 4h) and CD40L expression (P<0.05 VS control at 2h and 4h). Coffee drinking also inhibited arachidonic acid induced GPⅡb/Ⅲa (P<0.05 VS control at 2h and 4h) and CD40L expression (P<0.05 VS control at 2h). Caffeine intake did not affect platelet GPⅡb/Ⅲa, CD40L expression induced by the agonists.Conclusion These results indicate that coffee is active in inhibiting platelet GPⅡb/Ⅲa and CD40L expression to some extent in vivo, and may be responsible for the beneficial effects of platelet function. Caffeine dose not show such effect at safe concentrations.%目的 研究咖啡对血小板功能的影响,阐明其中可能的机制.方法 12名健康男性被随机分配到三组之一,进行为期3w的交叉试验,分别饮用相同咖啡因含量(180 mg)的咖啡、咖啡因片剂或安慰剂.分别在即刻0、2、4h采血,对血小板进行全血流式细胞仪分析,测定血小板活化状态糖蛋白ⅡbⅢa(glycoprotein ⅡbⅢa, GPⅡbⅢa)、CD40L表达.另在体外,测定不同浓度咖啡因对血小板GPⅡbⅢa影响.结果 与对照组比较,饮用咖啡抑制二磷酸腺苷(ADP)诱导的GPⅡbⅢa表达(2h和4h,P <0.05)和CD40L表达(2h和4h,P <0.05).咖啡也抑制花生四烯酸诱导GPⅡbⅢa表达(2h和4h,P <0.05)和CD40L表达(2h,P <0.05).安全剂量咖啡因的摄入不影响血小板活化后GPⅡbⅢa和CD40L表达.结论 咖啡在体内可以通

  14. The Relationship between CD40/CD40L Signal System and Preeclampsia%CD40/CD40L信号系统与子痫前期的关系

    Institute of Scientific and Technical Information of China (English)

    隋仁芳; 吴春凤; 孙敬霞; 黄福丹; 安牧尔

    2013-01-01

    子痫前期是病理产科中常见的疾病,是导致孕产妇和围生儿死亡的主要原因,其具体病因目前尚未完全阐明,大多数学者认为主要与氧化应激导致的血管内皮细胞损伤,胎盘浅着床,免疫平衡的破坏等因素有关,其中内皮细胞损伤导致的内皮细胞生理功能失衡是其病因学研究的热点.CD40/CD40L是一对互补的肿瘤坏死因子和肿瘤坏子因子受体超家族跨膜糖蛋白,相互作用可以促进内皮细胞、平滑肌细胞表达和释放细胞因子、组织因子、金属蛋白酶、粘附分子,这些物质间接或直接的参与了炎症反应和免疫调节,导致内皮细胞损伤.因此,CD40/CD40L相互作用在炎症反应和免疫失调中亦起着关键作用,可能是子痫前期的病因之一,以CD40/CD40L为靶点为子痫前期的诊断和治疗提供了新的思路和方向.本文拟对CD40/CD40L信号系统与子痫前期的关系做一综述.%Preeclampsia is a common pathological obstetrics disease and a major cause of maternal and perinatal morbidity and mortality. The etiology of pathological obstetrics disease is not very clear to us. Most scholars believe that it relates to the factors of vascular endothelial cell injury caused by Oxidative stress, placental shallow implantation, and destruction of immune balance, endothelial cell dysfunction caused by endothelial cell injury is hot issue of etiology research, CD40 ligand (CD40L; CD 154) and its receptor CD40, costimulatory molecules of the tumor necrosis factor (TNF) and TNF receptor family. Interaction can promote endothelial cell and smooth muscle cell expression and release cytokines,tissue factor, adhesion molecules and metalloproteinases, these substances directly or indirectly involved in modulating immune responses and inflammation, resulting in endothelial cell damage, and CD40/CD40L involved in occurrence and development of Atherosclerosis, CD40/CD40L is considered as one possible etiology of

  15. Induction of IL-12 Production in Human Peripheral Monocytes by Trypanosoma cruzi Is Mediated by Glycosylphosphatidylinositol-Anchored Mucin-Like Glycoproteins and Potentiated by IFN-γ and CD40-CD40L Interactions

    Directory of Open Access Journals (Sweden)

    Lúcia Cristina Jamli Abel

    2014-01-01

    Full Text Available Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi, is characterized by immunopathology driven by IFN-γ secreting Th1-like T cells. T. cruzi has a thick coat of mucin-like glycoproteins covering its surface, which plays an important role in parasite invasion and host immunomodulation. It has been extensively described that T. cruzi or its products—like GPI anchors isolated from GPI-anchored mucins from the trypomastigote life cycle stage (tGPI-mucins—are potent inducers of proinflammatory responses (i.e., cytokines and NO production by IFN-γ primed murine macrophages. However, little is known about whether T. cruzi or GPI-mucins exert a similar action in human cells. We therefore decided to further investigate the in vitro cytokine production profile from human mononuclear cells from uninfected donors exposed to T. cruzi as well as tGPI-mucins. We observed that both living T. cruzi trypomastigotes and tGPI-mucins are potent inducers of IL-12 by human peripheral blood monocytes and this effect depends on CD40-CD40L interaction and IFN-γ. Our findings suggest that the polarized T1-type cytokine profile seen in T. cruzi infected patients might be a long-term effect of IL-12 production induced by lifelong exposure to T. cruzi tGPI-mucins.

  16. Cell enrichment-free massive ex-vivo expansion of peripheral CD20⁺ B cells via CD40-CD40L signals in non-human primates.

    Science.gov (United States)

    Kim, Jung-Sik; Byun, Nari; Chung, Hyunwoo; Kim, Hyun-Je; Kim, Jong-Min; Chun, Taehoon; Lee, Won-Woo; Park, Chung-Gyu

    2016-04-22

    Non-human primates (NHPs) are valuable as preclinical resources that bridge the gap between basic science and clinical application. B cells from NHPs have been utilized for the development of B-cell targeted drugs and cell-based therapeutic modalities; however, few studies on the ex-vivo expansion of monkey B cells have been reported. In this study, we developed a highly efficient ex-vivo expansion protocol for monkey B cells resulting in 99% purity without the requirement for prior cell-enrichment procedures. To this end, monkey peripheral blood mononuclear cells (PBMCs) were stimulated for 12 days with cells constitutively expressing monkey CD40L in expansion medium optimized for specific and massive expansion of B cells. The B cells expansion rates obtained were 2-5 times higher than those previously reported in humans, with rates ranging from 7.9 to 16.6 fold increase. Moreover, expanded B cells sustained high expression of co-stimulatory molecules including CD83 and CD86 until day 12 of culture, and the simple application of a brief centrifugation resulted in a CD20(+) B cell purity rate of greater than 99%. Furthermore, small amounts of CD3(+)CD20(+)BT-like cells were generated and CD16 was expressed at moderate levels on expanded B cells. Thus, the establishment of this protocol provides a method to produce quantities of homogeneous, mature B cells in numbers sufficient for the in vitro study of B cell immunity as well as for the development of B cell-diagnostic tools and cell-based therapeutic modalities.

  17. The Expression of CD40/CD40L and OX40 in Acute Myeloid Leukaemia%CD40-CD40L与OX40在急性髓性白血病中的表达意义

    Institute of Scientific and Technical Information of China (English)

    师帅帅; 乔振华

    2009-01-01

    目的:检测在急性髓性白血病细胞中CD40、CD40L、OX40的表达并探讨其临床意义.方法:应用流式细胞仪检测46例未经治疗的初诊急性髓性白血病患者骨髓单个核细胞表面CD40、CD40L、OX40的表达,分为四组:仅表达CD40的患者组(①组)、表达CD40+CD40L+OX40-的患者组(②组)、三种抗原全部表达组(③组)和全部不表达组(④组).利用SPSS统计软件分析.结果:在有高白细胞表现的患者中,②组的发病率(100%)最高;在有血小板减少表现的患者中,①组的发病率(100%)最高;在有脾大表现的患者中,④组的发病率(6.7%)最低.结论:CD40的单独表达可能使白血病患者血小板的降低更加严重,而当合并CD40L表达时,会减少血小板低下的发生率.CD40的表达能增加患者伴有脾大表现的可能性.

  18. Th cells promote CTL survival and memory via acquired pMHC-I and endogenous IL-2 and CD40L signaling and by modulating apoptosis-controlling pathways.

    Directory of Open Access Journals (Sweden)

    Channakeshava Sokke Umeshappa

    Full Text Available Involvement of CD4(+ helper T (Th cells is crucial for CD8(+ cytotoxic T lymphocyte (CTL-mediated immunity. However, CD4(+ Th's signals that govern CTL survival and functional memory are still not completely understood. In this study, we assessed the role of CD4(+ Th cells with acquired antigen-presenting machineries in determining CTL fates. We utilized an adoptive co-transfer into CD4(+ T cell-sufficient or -deficient mice of OTI CTLs and OTII Th cells or Th cells with various gene deficiencies pre-stimulated in vitro by ovalbumin (OVA-pulsed dendritic cell (DCova. CTL survival was kinetically assessed in these mice using FITC-anti-CD8 and PE-H-2K(b/OVA257-264 tetramer staining by flow cytometry. We show that by acting via endogenous CD40L and IL-2, and acquired peptide-MHC-I (pMHC-I complex signaling, CD4(+ Th cells enhance survival of transferred effector CTLs and their differentiation into the functional memory CTLs capable of protecting against highly-metastasizing tumor challenge. Moreover, RT-PCR, flow cytometry and Western blot analysis demonstrate that increased survival of CD4(+ Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2 and down-regulation of proapoptotic (Bcl-10, Casp-3, Casp-4, Casp-7 molecules. Taken together, our results reveal a previously unexplored mechanistic role for CD4(+ Th cells in programming CTL survival and memory recall responses. This knowledge could also aid in the development of efficient adoptive CTL cancer therapy.

  19. 丹红注射液对急性脑梗死患者sCD40L及超敏C-反应蛋白的影响%Efficacy of Danhong injection for patients with acute stage of cerebral infarction and its effects on serum sCD40L and hs-CRP levels

    Institute of Scientific and Technical Information of China (English)

    沈壮虹; 宋水江

    2013-01-01

    Objective To evaluate efficacy of Danhong injection for patients with acute stage of cerebral infarction and its effects on serum levels of soluble CD40 ligand (sCD40L) and high-sensitivity C-reactive protein(hs-CRP). Methods Ninety two patients with acute ischemic cerebral infarction were randomly assigned to receive Danhong injection (30ml/d, i.v drip, for 14d) in addition to routine treatment (Danhong group) or routine treatment only(control group).Serum sCD40L and hs-CRP were mea-sured, and Barthel index (BI) and Chinese stroke Scale (CSS) were assessed before and after treatment in both groups. Results The levels of sCD40L and hs-CRP were deceased after treatment in both groups, however, the levels in Danhong group were significantly lower than those in control group (P<0.05). The scores of CSS and BI of two groups were improved after treatment, while scores in Danhong group were better than those in control group (P<0.05). Conclusion Danhong injection can decrease serum levels of sCD40L and hs-CRP and improve the recovery of neural function for patients with acute cerebral infarction.%  目的探讨步长丹红注射液对急性脑梗死患者血清可溶性白细胞分化抗原40配体(sCD40L)及超敏 C-反应蛋白(hs-CRP)的影响及临床疗效。方法92例急性脑梗死患者分为两组,即常规治疗组和丹红治疗组,丹红治疗组在常规治疗基础上给予步长丹红注射液30ml 溶于0.9%氯化钠注射液250ml,1次/d,静脉滴注,共14d。治疗前后测定血 sCD40L 及 hs-CRP 的变化,并用中国卒中量表(CSS)及巴氏指数(BI)评定。结果两组患者 sCD40L 及 hs-CRP 均下降,丹红治疗组均明显低于常规治疗组(均 P<0.05)。治疗后两组 CSS 及 BI 评分较治疗前均明显增加,丹红治疗组均高于常规治疗组(均 P<0.05)。结论步长丹红注射液可降低急性脑梗死患者 sCD40L 及 hs-CRP 的水平,有益于患者神经功能的恢复。

  20. 血清可溶性CD40L和C反应蛋白在慢性血透患者外周血管钙化的作用%Role of soluble CD40L and C-reactive protein in peripheral vascular calcification in patients undergoing of chronic hemodialysis

    Institute of Scientific and Technical Information of China (English)

    伍强; 孙艳; 杨铁城; 张海燕

    2010-01-01

    Objective To investigate the role of soluble CD40 ligand (sCD40L) and C-reactive protein (CRP) in peripheral vascular calcification in hemodialytic patients.Methods 40 hemodialytic patients with uremia underwent arteriovenous fistula reconstruction.The stumps of radial artery were removed for assessing vascular calcification.The patients were grouped according to the presence or absence of calcification and its degree.Serum levels of sCD40L and CRP were detected.The differences in levels of sCD40L and CRP were compared between calcification group and non-calcification group and between calcification groups at different degrees calcification.Results Radial artery calcification was detectable in 23 of the patients with calcium staining,9 of whom had severe calcification and the rest 14 had mild to moderate calcification.Levels of sCD40L and CRP were significantly higher in calcification group than in non-calcification group (P<0.01); they were also higher in severe calcification group than in mild to moderate calcification group (P<0.05).Levels of sCD40L were positively correlated with those of CRP.%目的 探讨血清可溶性白细胞分化抗原40配体(sCD40L)和C反应蛋白(CRP)在尿毒症血液透析患者外周血管钙化中的作用.方法 尿毒症血液透析患者40例因动静脉内瘘失功行内瘘重建术,术中取桡动脉残端行钙化染色观察血管钙化情况,依据血管钙化有无及程度分组,同时检测血清sCD40L和CRP水平,比较钙化组与非钙化组,不同程度钙化组间血清sCD40L和CRP水平的差异.结果 40例患者桡动脉钙化染色检出血管钙化23例,其中重度钙化9例,轻中度钙化14例,钙化组血清sCD40L和CRP水平高于非钙化组,有统计学差异(P<0.01).重度钙化组sCD40L和CRP水平高于轻中度钙化组.有统计学差异(P<0.05).血清sCD40L和CRP含量呈正相关.结论 sCD40L和炎症反应可能参与了尿毒症血液透析患者外周血管钙化的发生.

  1. Changes of sCD40L and CRP in patients with cerebral infarction and its clinical significance%脑梗死患者sCD40L与CRP含量的改变及其临床意义

    Institute of Scientific and Technical Information of China (English)

    凌启节; 储照虎

    2012-01-01

    目的 探讨sCD40L和CRP的含量与动脉粥样硬化性脑梗死(Atherosclerotic cerebral infarction,ACI)面积、NIHSS量表评分之间的相关性.方法 采用双抗体夹心酶联免疫法及比浊法检测63例ACI患者及30例体检健康者的血清sCD40L和CRP含量,采用SPSS16.0软件进行统计学分析.结果 ACI患者血清sCD40L和CRP含量显著高于正常对照组,且病情越重及脑梗死面积越大的患者,血清sCD40L和CRP含量就越高,入院即时和入院14 d血清sCD40L与CRP含量呈正相关,通过绘制ROC曲线得出,sCD40L曲线下面积(0.954)大于CRP曲线下面积(0.869),sCD40L特异度(100%)高于CRP(96.7%),sCD40L灵敏度(84.1%)也高于CRP(69.8%).结论 sCD40L、CRP含量的改变可作为判断ACI病情的血清生物学指标.%Objective To investigate the correlation between the content of sCD4()L and CRP and atherosclerotic cerebral infarction area, NIHSS scale score. Methods Scrum sCD4()L and CRP levels of 63 ACI patients and 30 healthy persons were tested by double antibody sandwich enzyme immunoassay and turbidimctric assay, SPSS 16 software was used for statistical analysis. Results ACI patients with scrum sCD4()L and CRP contents were significantly higher than in normal controls, and more advanced disease and cerebral infarction area more patients, the scrum sCD4()L and CRP content is higher, the instant and the 14 day of admission admission scrum sCD4()L and CRP levels were positively associated, by plotting ROC curves, the area under the sCD4()L curve (0. 954) is greater than CRP curve area (0. 869) , sCD4()L specificity (100%) was higher than that of CRP (96. 7%), sCD40L sensitivity (84. 1 %) over CRP (69. 8%). Conclusions SCD40L, CRP content change can be used to judge the ACI disease scrum biological markers.

  2. 血清Hcy、sCD40L及LDL-C与冠状动脉支架内再狭窄的相关性研究%Postprocedural serum Hcy, sCD40L and LDL-C are associated with coronary in-stent restenosis in patients with drug-coated stent implantation

    Institute of Scientific and Technical Information of China (English)

    陈玉映; 胡允兆; 陈盈文; 何宗云; 郑素琳; 吴焱贤

    2012-01-01

    目的:探讨血清同型半胱氨酸(Hcy)、LDL-C、可溶性白细胞表面抗原40配体(sCD40L)等因素在冠状动脉支架内再狭窄发生过程中的临床意义.方法:收集冠状动脉药物涂层支架植入术后因胸痛再发/术后6个月常规行冠状动脉造影随访患者共120例,其中88例造影未出现支架内再狭窄(对照组),32例出现再狭窄(再狭窄组),回顾分析两组患者的年龄、性别、高血压病史、糖尿病史、心肌梗死病史及冠心痛家族史,收集患者支架植入术后1周以及胸痛再发/术后6个月冠状动脉造影检查这两个时间点的BMI、肾小球滤过率(GFP)、甘油三酯、总胆固醇、糖化血红蛋白( GHbA1c)、LDL-C、HDL-C、Hcy、sCD40L等指标.结果:两组患者在支架植入术后1周各指标比较差异无统计学意义,因胸痛再发/术后6个月行冠状动脉造影随访时发现再狭窄组的LDL-C、Hey、sCD40L和对照组比较差异有统计学意义(P<0.01),二分类变量Logistic回归分析显示,LDL-C、sCD40L及Hcy的升高会增加再狭窄的风险,且Hcy与LDL-C(r=0.36,P=0.002)、sCD40L(r=0.27,P=0.001)呈正相关.结论:支架植入术后LDL-C、Hcy、sCD40L升高是支架内再狭窄的危险因子,并且Hcy与LDL-C、sCD40L呈正相关.%Objective: To evaluate the roles of serum homocysteine ( Hey), low density lipoprotein cholesterol (LDL-C) and soluble leukocyte surface antigens 40 ligand (sCD40L) in in-stent restenosis (ISR) after successful primary percutaneous coronary intervention (PCI). Methods; Body mass index (BMI) , glomerular filtration rate (GFP) , total triglycerides (TG) , total cholesterol (TC) , glycosylated hemoglobin (GHbAlc) , LDL-C, high density lipoprotein ( HDL-C) , Hey and sCD40L levels were measured in 120 consecutive patients 1 week and 6 months ( or chest pain occurred) after PCI. These patients were grouped as ISR and non-ISR based on angiography during follow-up time. Besides, age, sex, hypertension, diabetes

  3. B-NHL患者骨髓单个核细胞表面CD40、CD40L的表达及意义%Expressions and significance of CD40,CD40L in bone marrow mononuclear cell surface of patients with B-cell Non-Hodgkin′s Lymphoma

    Institute of Scientific and Technical Information of China (English)

    刘爱春; 任宏伟; 鄂明艳; 王燕

    2013-01-01

    目的:探讨B细胞非霍奇金淋巴瘤(B cell Non-Hodgkin′s Lymphoma,B-NHL)患者骨髓单个核细胞( BMMC)表面CD40、 CD40 L的表达情况及变化规律,分析CD40、CD40 L表达与淋巴瘤发病、侵袭性及预后的关系及意义。方法采集20例初诊B-NHL伴有骨髓侵犯的患者(实验组)的骨髓标本及12例健康者(对照组)的骨髓标本,分离单个核细胞,采用直接荧光抗体标记,应用流式细胞仪检测细胞表面CD40、CD40 L的表达情况。相关实验结果进行统计学比较。结果(1) B-NHL中CD40阳性率(45.78±3.82)%显著高于正常对照组(13.99±2.20)%,P<0.01;B-NHL中CD40L阳性率(3.44±0.88)%显著低于正常对照组(10.64±1.03)%,P<0.01;(2) B -NHL 伴有骨髓侵犯的患者CD40、CD40L阳性率与病理类型、LDH值、全身症状呈显著相关,P<0.05;(3) B-NHL伴有骨髓侵犯的患者中CD40与CD40L表达呈负相关,r =-0.47,P<0.05。结论(1) CD40、CD40L阳性率与病理类型、LDH值、全身症状密切相关,其可能与B-NHL的侵袭性及预后有关;(2) B-NHL患者中CD40表达的增高、CD40L表达的减少可能是影响其发病的因素之一;(3) B-NHL患者中CD40L表达的减少可能与肿瘤免疫逃逸机制有关。%Objective To explore the expressions and changing regularity of CD 40,CD40L in surface of bone marrow mononuclear cell (BMMC)of patients with B -cell Non-Hodgkin′s Lymphoma.To study the rela-tionship between the expressions of CD 40,CD40L and the pathogenesis、infestation、prognosis,clinical implication of patients with Lymphoma .Methods Twenty bone marrow specimens of B -NHL ( experimental group ) and twelve healthy bone marrow specimens(contiol group)were collected.To separate mononuclear cell,we used di-rect fluorescent antibody to mark cells ,the expressions of CD40 and CD40L were determined by flow cytometry

  4. Complexes between nuclear factor-κB p65 and signal transducer and activator of transcription 3 are key actors in inducing activation-induced cytidine deaminase expression and immunoglobulin A production in CD40L plus interleukin-10-treated human blood B cells.

    Science.gov (United States)

    Lafarge, S; Hamzeh-Cognasse, H; Richard, Y; Pozzetto, B; Cogné, M; Cognasse, F; Garraud, O

    2011-11-01

    The signal transducer and activator of transcription 3 (STAT3) transcription factor pathway plays an important role in many biological phenomena. STAT3 transcription is triggered by cytokine-associated signals. Here, we use isolated human B cells to analyse the role of STAT3 in interleukin (IL)-10 induced terminal B cell differentiation and in immunoglobulin (Ig)A production as a characteristic readout of IL-10 signalling. We identified optimal conditions for inducing in-vitro IgA production by purified blood naive B cells using IL-10 and soluble CD40L. We show that soluble CD40L consistently induces the phosphorylation of nuclear factor (NF)-κB p65 but not of STAT3, while IL-10 induces the phosphorylation of STAT3 but not of NF-κB p65. Interestingly, while soluble CD40L and IL-10 were synergistic in driving the terminal maturation of B cells into IgA-producing plasma cells, they did not co-operate earlier in the pathway with regard to the transcription factors NF-κB p65 or STAT3. Blocking either NF-κB p65 or STAT3 profoundly altered the production of IgA and mRNA for activation-induced cytidine deaminase (AID), an enzyme strictly necessary for Ig heavy chain recombination. Finally, the STAT3 pathway was directly activated by IL-10, while IL-6, the main cytokine otherwise known for activating the STAT3 pathway, did not appear to be involved in IL-10-induced-STAT3 activation. Our results suggest that STAT3 and NF-κB pathways co-operate in IgA production, with soluble CD40L rapidly activating the NF-κB pathway, probably rendering STAT3 probably more reactive to IL-10 signalling. This novel role for STAT3 in B cell development reveals a potential therapeutic or vaccine target for eliciting IgA humoral responses at mucosal interfaces.

  5. DNA Profiling of Convicted Offender Samples for the Combined DNA Index System

    Science.gov (United States)

    Millard, Julie T

    2011-01-01

    The cornerstone of forensic chemistry is that a perpetrator inevitably leaves trace evidence at a crime scene. One important type of evidence is DNA, which has been instrumental in both the implication and exoneration of thousands of suspects in a wide range of crimes. The Combined DNA Index System (CODIS), a network of DNA databases, provides…

  6. Changes of levels of plasma sCD40L,MMP-9 and TIMP-1 in patients with acute coronary syndrome and their clinical significance%急性冠脉综合征患者血浆sCD40L与血清基质金属蛋白酶水平的变化及其意义

    Institute of Scientific and Technical Information of China (English)

    赵晓辉; 王皓娟; 李淑红; 牟春平

    2011-01-01

    Objective: To investigate changes of levels of soluble CD40 ligand (sCD40L), serum matrix metalloprotein-ase—9 (MMP—9) and serum tissue inhibitor of metalloproteinases — 1 (TIMP— 1) in patients with acute coronary syndrome (ACS) and their correlation. Methods: Enzyme—linked immunosorbent assay was used to measure levels of sCD40L, MMP—9 and TIMP—1 in 70 patients with coronary heart disease (CHD) [35 ACS cases, 35 cases with stable angina pectoris (SAP)] and 35 non— CHD patients (normal control group). Results: Compared with normal control group and SAP group, the levels of sCD40L [ (2. 73±0. 92) μg/ml vs. (3. 05±0. 98) μg/ml vs. (4. 72±1. 15) pig/ml] and MMP-9 [ (152. 38±54. 22) ng/ml vs. (341. 12±69. 96) ng/ml vs. (574. 2±139. 20) ng/ml] significantly increased, and level of TIMP-1 [ (415. 92±13. 96) ng/ml vs. (249. 32±36. 80) ng/ml vs. (172. 20±40. 10) ng/ml] significantly decreased in ACS group, P<0. 01 all; MMP—9 level was positively correlated with sCD40L level (r=0. 42, P<0. 05). Conclusion: Increased levels of sCD40L and serum MMP—9 and decreased serum TIMP—1 level in ACS patients may be related with instability of atheromatous plaque, and they could serve as serological indicators for instability of atheromatous plaque.%目的:观察急性冠脉综合征(ACS)患者可溶性CD40配体(sCD40L)及血清基质金属蛋白酶-9(MMP-9)、血清组织金属蛋白酶抑制物-1(TIMP-1)水平变化及其相关性.方法:采用酶联免疫吸附法测定70例冠心病患者[ACS患者35例、稳定型心绞痛(SAP)患者35例]、35例非冠心病患者(正常对照组)sCD40L、MMP-9,TIMP-1的水平.结果:与正常对照组及SAP组比较,ACS组sCD40L[(2.73±0.92)μg/ml比(3.05±0.98)μg/ml比(4.72±1.15)μg/ml]、MMP-9[(152.38±54.22)ng/ml比(341.12±69.96)ng/ml比(574.2±139.20)ng/ml]水平明显升高(P均<0.01),而TIMP-1[(415.92±13.96)ng/ml比(249.32±36.80)ng/ml比(172.20±40.10)ng/ml]水平明显降低(P<0.01);且MMP-9

  7. Value of brachial artery FMD and sCD40L in predicting acute coronary syndrome%超声检查肱动脉FMD和sCD40L对急性冠状动脉综合征的预测价值

    Institute of Scientific and Technical Information of China (English)

    赵娟; 张小勇; 邓彩妹; 沈锡林; 潘伟

    2014-01-01

    目的:探讨超声检查肱动脉血流介导性舒张( FMD )和血清可溶性细胞分化抗原40配体(sCD40L)对急性冠状动脉综合征(ACS)的预测价值。方法随机入选39名健康志愿者、39例稳定性心绞痛患者(SA组)和79例急性冠状动脉综合征(ACS)患者。 ACS患者包括不稳定性心绞痛患者49例(UA组),急性心肌梗死患者30例(AMI组)。用超声检查肱动脉FMD,并用ELISA法检测血清sCD40L的含量。研究ACS患者肱动脉FMD和血清sCD40L的变化。用Pearson相关性分析ACS患者肱动脉FMD与血清sCD40L水平的关系。结果肱动脉 FMD 正常对照组(9.52±2.54)%, SA 组(5.35±1.24)%, UA 组(3.32±1.41)%,AMI组(3.26±1.32)%,ACS患者肱动脉FMD下调(P<0.05)。血清sCD40L正常对照组(1.21±0.31)ng/ml,SA组(4.12±0.98)ng/ml,UA组(12.86±2.67)ng/ml,AMI组(13.95±2.83)ng/ml,ACS患者血清sCD40L升高(P<0.05)。 ACS患者肱动脉FMD与血清sCD40L水平呈负相关,r=-0.708,P=0.000。结论超声发现肱动脉FMD下调,血清sCD40L升高,对ACS有预测价值。%Objective To investigate the predictive value of ACS by brachial artery FMD and sCD 40L. Methods Thirty-nine healthy volunteers(control group), 39 patients with stable angina pectoris(SA group) and 79 patients with acute coronary syndrome (ACS),including 49 patients with unstable angina pectoris (UA group) and 30 patients with acute myocardial infarction (AMI group), were radomly selected.The patients with ACS were detected the brachial artery FMD by ultrasonic testing and the serum sCD 40L by ELISA.Relation of brachial artery FMD and serum sCD40L of ACS patients were analyzed by using Pearson correlation analysis .Results Compared with control group and SA goup , the brachial artery FMD decreased in UA group and AMI group ( 9.52 ±2.54 )%( control group) vs (5.35 ±1.24)%(SA group) vs (3.32 ±1.41)% (UA group) vs (3

  8. Novel Vaccine against Venezuelan Equine Encephalitis Combines Advantages of DNA Immunization and a Live Attenuated Vaccine

    OpenAIRE

    Tretyakova, Irina; Lukashevich, Igor S; Glass, Pamela; Wang, Eryu; Weaver, Scott; Pushko, Peter

    2012-01-01

    DNA vaccines combine remarkable genetic and chemical stability with proven safety and efficacy in animal models, while remaining less immunogenic in humans. In contrast, live-attenuated vaccines have the advantage of inducing rapid, robust, long-term immunity after a single-dose vaccination. Here we describe novel iDNA vaccine technology that is based on an infectious DNA platform and combines advantages of DNA and live attenuated vaccines. We applied this technology for vaccination against i...

  9. Combining optical tweezers and scanning probe microscopy to study DNA-protein interactions

    NARCIS (Netherlands)

    Huisstede, Jurgen H.G.; Subramaniam, Vinod; Bennink, Martin L.

    2007-01-01

    We present the first results obtained with a new instrument designed and built to study DNA-protein interactions at the single molecule level. This microscope combines optical tweezers with scanning probe microscopy and allows us to locate DNA-binding proteins on a single suspended DNA molecule. A s

  10. Determination of chromium combined with DNA, RNA and protein in chromium-rich brewer's yeast

    International Nuclear Information System (INIS)

    The contents of chromium in the DNA, RNA and protein fractions separated from chromium-rich and normal brewer's yeast were determined with the neutron activation analysis in order to study the combination of Cr with DNA, RNA and protein in chromium-rich brewer's yeast. The results showed that the extracting rats and concentrations of DNA, RNA and protein had no significant difference in two types of yeast, but the chromium contents of DNA, RNA and protein in the chromium-rich yeast were significantly higher than those in the normal. In addition, the content of chromium in DNA was much higher than that in RNA and protein, which indicated that the inorganic chromium compounds entered into the yeast cell, during the yeast cultivation in the culture medium containing chromium were converted into organic chromium compounds combined with DNA, RNA and protein

  11. A combined DNA vaccine provides protective immunity against Mycobacterium bovis and Brucella abortus in cattle.

    Science.gov (United States)

    Hu, Xi-Dan; Yu, Da-Hai; Chen, Su-Ting; Li, Shu-Xia; Cai, Hong

    2009-04-01

    We evaluated the immunogenicity and protective efficacy of a combined DNA vaccine containing six genes encoding immunodominant antigens from Mycobacterium bovis and Brucella abortus. The number of lymph node and spleen cultures positive for M. bovis and B. abortus from calves immunized with the combined DNA vaccine was significantly reduced (p abortus 544. The combined DNA vaccine group displayed stronger antigen-specific interferon-gamma (IFN-gamma) responses and antigen-specific IFN-gamma ELISPOT activities 2 months after final immunization and after challenge. Antigen-specific CD4(+) and CD8(+) T cell responses in the combined DNA vaccine group were higher than either the Bacillus Calmette-Guerin (BCG)-positive or S19-positive control group. Likewise, more calves in the DNA vaccine group exhibited antigen-specific IgG titers and had higher IgG titers than those in the BCG- or S19-immunized groups 2 months after the final immunization. Moreover, two antigens in the combined DNA vaccine induced significant antigen-specific IFN-gamma responses 6 months after challenge (p S19 against B. abortus. This is the first report to demonstrate that a single combined DNA vaccine protects cattle against two infectious diseases. PMID:19364278

  12. A combined HM-PCR/SNuPE method for high sensitive detection of rare DNA methylation

    Directory of Open Access Journals (Sweden)

    Tierling Sascha

    2010-06-01

    Full Text Available Abstract Background DNA methylation changes are widely used as early molecular markers in cancer detection. Sensitive detection and classification of rare methylation changes in DNA extracted from circulating body fluids or complex tissue samples is crucial for the understanding of tumor etiology, clinical diagnosis and treatment. In this paper, we describe a combined method to monitor the presence of methylated tumor DNA in an excess of unmethylated background DNA of non-tumorous cells. The method combines heavy methyl-PCR, which favors preferential amplification of methylated marker sequence from bisulfite-treated DNA with a methylation-specific single nucleotide primer extension monitored by ion-pair, reversed-phase, high-performance liquid chromatography separation. Results This combined method allows detection of 14 pg (that is, four to five genomic copies of methylated chromosomal DNA in a 2000-fold excess (that is, 50 ng of unmethylated chromosomal background, with an analytical sensitivity of > 90%. We outline a detailed protocol for the combined assay on two examples of known cancer markers (SEPT9 and TMEFF2 and discuss general aspects of assay design and data interpretation. Finally, we provide an application example for rapid testing on tumor methylation in plasma DNA derived from a small cohort of patients with colorectal cancer. Conclusion The method allows unambiguous detection of rare DNA methylation, for example in body fluid or DNA isolates from cells or tissues, with very high sensitivity and accuracy. The application combines standard technologies and can easily be adapted to any target region of interest. It does not require costly reagents and can be used for routine screening of many samples.

  13. Efficient vaccine against pandemic influenza: combining DNA vaccination and targeted delivery to MHC class II molecules.

    Science.gov (United States)

    Grødeland, Gunnveig; Bogen, Bjarne

    2015-06-01

    There are two major limitations to vaccine preparedness in the event of devastating influenza pandemics: the time needed to generate a vaccine and rapid generation of sufficient amounts. DNA vaccination could represent a solution to these problems, but efficacy needs to be enhanced. In a separate line of research, it has been established that targeting of vaccine molecules to antigen-presenting cells enhances immune responses. We have combined the two principles by constructing DNA vaccines that encode bivalent fusion proteins; these target hemagglutinin to MHC class II molecules on antigen-presenting cells. Such DNA vaccines rapidly induce hemagglutinin-specific antibodies and T cell responses in immunized mice. Responses are long-lasting and protect mice against challenge with influenza virus. In a pandemic situation, targeted DNA vaccines could be produced and tested within a month. The novel DNA vaccines could represent a solution to pandemic preparedness in the advent of novel influenza pandemics.

  14. DNA damage in peripheral blood lymphocytes in patients during combined chemotherapy for breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Sanchez-Suarez, Patricia [Oncological Research Unit, Oncology Hospital, National Medical Center S-XXI, Instituto Mexicano del Seguro Social (IMSS), Av. Cuauhtemoc 330, Col. Doctores, 06725 Mexico, D.F. (Mexico); Ostrosky-Wegman, Patricia [Biomedical Research Institute, Universidad Nacional Autonoma de Mexico (UNAM), Mexico City (Mexico); Gallegos-Hernandez, Francisco [Department of Clinical Oncology, Oncology Hospital, National Medical Center S-XXI, Instituto Mexicano del Seguro Social (IMSS), Mexico City (Mexico); Penarroja-Flores, Rubicelia; Toledo-Garcia, Jorge [Oncological Research Unit, Oncology Hospital, National Medical Center S-XXI, Instituto Mexicano del Seguro Social (IMSS), Av. Cuauhtemoc 330, Col. Doctores, 06725 Mexico, D.F. (Mexico); Bravo, Jose Luis [Atmospheric Sciences Institute, Universidad Nacional Autonoma de Mexico (UNAM), Mexico City (Mexico); Rojas del Castillo, Emilio [Biomedical Research Institute, Universidad Nacional Autonoma de Mexico (UNAM), Mexico City (Mexico); Benitez-Bribiesca, Luis [Oncological Research Unit, Oncology Hospital, National Medical Center S-XXI, Instituto Mexicano del Seguro Social (IMSS), Av. Cuauhtemoc 330, Col. Doctores, 06725 Mexico, D.F. (Mexico)], E-mail: luisbenbri@mexis.com

    2008-04-02

    Combined chemotherapy is used for the treatment of a number of malignancies such as breast cancer. The target of these antineoplastic agents is nuclear DNA, although it is not restricted to malignant cells. The aim of the present study was to assess DNA damage in peripheral blood lymphocytes (PBLs) of breast cancer patients subjected to combined adjuvant chemotherapy (5-fluorouracil, epirubicin and cyclophosphamide, FEC), using a modified comet assay to detect DNA single-strand breaks (SSB) and double-strand breaks (DSB). Forty-one female patients with advanced breast cancer before and after chemotherapy and 60 healthy females participated in the study. Alkaline and neutral comet assays were performed in PBLs according to a standard protocol, and DNA tail moment was measured by a computer-based image analysis system. Breast cancer patients before treatment had higher increased background levels of SSB and DSB as compared to healthy women. During treatment, a significant increase in DNA damage was observed after the 2nd cycle, which persisted until the end of treatment. Eighty days after the end of treatment the percentage of PBLs with SSB and DSB remained elevated, but the magnitude of DNA damage (tail moment) returned to baseline levels. There was no correlation between PBL DNA damage and response to chemotherapy. DNA-SSB and DSB in PBLs are present in cancer patients before treatment and increase significantly after combined chemotherapy. No correlation with response to adjuvant chemotherapy was found. Biomonitoring DNA damage in PBLs of cancer patients could help prevent secondary effects and the potential risks of developing secondary cancers.

  15. COMPARISON OF DIFFERENT ENZYMES AND PROBES AND THEIR COMBINATIONS IN DNA FINGERPRINTING

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In the present study, eight combinations of restriction enzymes and oligonucleotide probes were tested for detecting VNTR polymorphism. More than a hundred loci were detected by all enzyme-probe combi nations. The influences of breed, enzyme and probe as well as their interactions were analysed, and the mean value of DNA fingerprint data was calculated for the enzymes and probes. The results will provide some valu- able information for studying the genetic relationship of individuals or populations using DNA fingerprinting.

  16. Combining crystallography and EPR: crystal and solution structures of the multidomain cochaperone DnaJ

    Energy Technology Data Exchange (ETDEWEB)

    Barends, Thomas R. M., E-mail: thomas.barends@mpimf-heidelberg.mpg.de [MPI for Medical Research, Heidelberg (Germany); Brosi, Richard W. W. [Freie Universitat Berlin, Berlin (Germany); Steinmetz, Andrea; Scherer, Anna; Hartmann, Elisabeth; Eschenbach, Jessica; Lorenz, Thorsten [MPI for Medical Research, Heidelberg (Germany); Seidel, Ralf [MPI for Molecular Physiology, Dortmund (Germany); Shoeman, Robert L.; Zimmermann, Sabine [MPI for Medical Research, Heidelberg (Germany); Bittl, Robert [Freie Universitat Berlin, Berlin (Germany); Schlichting, Ilme; Reinstein, Jochen [MPI for Medical Research, Heidelberg (Germany)

    2013-08-01

    The crystal structure of the N-terminal part of T. thermophilus DnaJ unexpectedly showed an ordered GF domain and guided the design of a construct enabling the first structure determination of a complete DnaJ cochaperone molecule. By combining the crystal structures with spin-labelling EPR and cross-linking in solution, a dynamic view of this flexible molecule was developed. Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from Thermus thermophilus comprising the J, GF and C-terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure-based EPR spin-labelling studies as well as cross-linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ.

  17. Combining crystallography and EPR: crystal and solution structures of the multidomain cochaperone DnaJ

    International Nuclear Information System (INIS)

    The crystal structure of the N-terminal part of T. thermophilus DnaJ unexpectedly showed an ordered GF domain and guided the design of a construct enabling the first structure determination of a complete DnaJ cochaperone molecule. By combining the crystal structures with spin-labelling EPR and cross-linking in solution, a dynamic view of this flexible molecule was developed. Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from Thermus thermophilus comprising the J, GF and C-terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure-based EPR spin-labelling studies as well as cross-linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ

  18. Low-density lipoprotein peptide-combined DNA nanocomplex as an efficient anticancer drug delivery vehicle.

    Science.gov (United States)

    Zhang, Nan; Tao, Jun; Hua, Haiying; Sun, Pengchao; Zhao, Yongxing

    2015-08-01

    DNA is a type of potential biomaterials for drug delivery due to its nanoscale geometry, loading capacity of therapeutics, biocompatibility, and biodegradability. Unfortunately, DNA is easily degraded by DNases in the body circulation and has low intracellular uptake. In the present study, we selected three cationic polymers polyethylenimine (PEI), hexadecyl trimethyl ammonium bromide (CTAB), and low-density lipoprotein (LDL) receptor targeted peptide (RLT), to modify DNA and improve the issues. A potent anti-tumor anthracycline-doxorubicin (DOX) was intercalated into DNA non-covalently and the DOX/DNA was then combined with PEI, CTAB, and RLT, respectively. Compact nanocomplexes were formed by electrostatic interaction and could potentially protect DNA from DNases. More importantly, RLT had the potential to enhance intracellular uptake by LDL receptor mediated endocytosis. In a series of in vitro experiments, RLT complexed DNA enhanced intracellular delivery of DOX, increased tumor cell death and intracellular ROS production, and reduced intracellular elimination of DOX. All results suggested that the easily prepared and targeted RLT/DNA nanocomplexes had great potential to be developed into a formulation for doxorubicin with enhanced anti-tumor activity.

  19. [Combined hopping-superexchange mechanism of charge transfer in DNA; a model with nearest interactions].

    Science.gov (United States)

    Lakhno, V D; Sultanov, V B

    2007-01-01

    In the framework of the earlier developed combined hopping-superexchange mechanism of charge transfer in DNA, a model with all nearest interactions between nucleobases is proposed. It is shown that the transfer rates for various types of nucleotide sequences calculated within this model are in a good agreement with experimental data.

  20. Synergistic antitumor efficacy of combined DNA vaccines targeting tumor cells and angiogenesis.

    Science.gov (United States)

    Yin, Xiaotao; Wang, Wei; Zhu, Xiaoming; Wang, Yu; Wu, Shuai; Wang, Zicheng; Wang, Lin; Du, Zhiyan; Gao, Jiangping; Yu, Jiyun

    2015-09-18

    To further enhance the antitumor efficacy of DNA vaccine, we proposed a synergistic strategy that targeted tumor cells and angiogenesis simultaneously. In this study, a Semliki Forest Virus (SFV) replicon DNA vaccine expressing 1-4 domains of murine VEGFR2 and IL12 was constructed, and was named pSVK-VEGFR2-GFc-IL12 (CAVE). The expression of VEGFR2 antigen and IL12 adjuvant molecule in 293T cells in vitro were verified by western blot and enzyme-linked immune sorbent assay (ELISA). Then CAVE was co-immunized with CAVA, a SFV replicon DNA vaccine targeting survivin and β-hCG antigens constructed previously. The antitumor efficacy of our combined replicon vaccines was evaluated in mice model and the possible mechanism was further investigated. The combined vaccines could elicit efficient humoral and cellular immune responses against survivin, β-hCG and VEGFR2 simultaneously. Compared with CAVE or CAVA vaccine alone, the combined vaccines inhibited the tumor growth and improved the survival rate in B16 melanoma mice model more effectively. Furthermore, the intratumoral microvessel density was lowest in combined vaccines group than CAVE or CAVA alone group. Therefore, this synergistic strategy of DNA vaccines for tumor treatment results in an increased antitumor efficacy, and may be more suitable for translation to future research and clinic. PMID:26253468

  1. Synergic Effect of Genistein and Daidzein on UVB-Induced DNA Damage: An Effective Photoprotective Combination

    Directory of Open Access Journals (Sweden)

    Barbara Iovine

    2011-01-01

    Full Text Available The anti-inflammatory effects and antioxidant activities of individual isoflavones are well established although little is known about the photoprotective effect of their combination. The aim of this study was to investigate the photoprotective effects of different concentrations of genistein and daidzein individually or combined. We measured the expression levels of the cyclo-oxygenase-2 (COX-2 and growth arrest and DNA-damage inducible (Gadd45 genes, which are involved in inflammation and DNA repair, respectively, in BJ-5ta human skin fibroblasts irradiated with 60 mJ/cm2 UVB. We also determined the cellular response to UVB-induced DNA damage by Comet assay. We report that genistein and daidzein when administered combined, and at a specific concentration and ratio, exerted a synergistic photoprotective effect that was greater than the effect obtained with each isoflavone alone. The results reported herein suggest that low concentrations of genistein and daidzein combined may be good candidate ingredients for protective agents against UV-induced photodamage.

  2. Dual Targeting Biomimetic Liposomes for Paclitaxel/DNA Combination Cancer Treatment

    Directory of Open Access Journals (Sweden)

    Guo-Xia Liu

    2014-08-01

    Full Text Available Combinations of chemotherapeutic drugs with nucleic acid has shown great promise in cancer therapy. In the present study, paclitaxel (PTX and DNA were co-loaded in the hyaluronic acid (HA and folate (FA-modified liposomes (HA/FA/PPD, to obtain the dual targeting biomimetic nanovector. The prepared HA/FA/PPD exhibited nanosized structure and narrow size distributions (247.4 ± 4.2 nm with appropriate negative charge of −25.40 ± 2.7 mV. HA/FA/PD (PTX free HA/FA/PPD showed almost no toxicity on murine malignant melanoma cell line (B16 and human hepatocellular carcinoma cell line (HepG2 (higher than 80% cell viability, demonstrating the safety of the blank nanovector. In comparison with the FA-modified PTX/DNA co-loaded liposomes (FA/PPD, HA/FA/PPD showed significant superiority in protecting the nanoparticles from aggregation in the presence of plasma and degradation by DNase I. Moreover, HA/FA/PPD could also significantly improve the transfection efficiency and cellular internalization rates on B16 cells comparing to that of FA/PPD (p < 0.05 and PPD (p < 0.01, demonstrating the great advantages of dual targeting properties. Furthermore, fluorescence microscope and flow cytometry results showed that PTX and DNA could be effectively co-delivered into the same tumor cell via HA/FA/PPD, contributing to PTX/DNA combination cancer treatment. In conclusion, the obtained HA/FA/PPD in the study could effectively target tumor cells, enhance transfection efficiency and subsequently achieve the co-delivery of PTX and DNA, displaying great potential for optimal combination therapy.

  3. Nanoparticles and DNA - a powerful and growing functional combination in bionanotechnology

    Science.gov (United States)

    Samanta, Anirban; Medintz, Igor L.

    2016-04-01

    Functionally integrating DNA and other nucleic acids with nanoparticles in all their different physicochemical forms has produced a rich variety of composite nanomaterials which, in many cases, display unique or augmented properties due to the synergistic activity of both components. These capabilities, in turn, are attracting greater attention from various research communities in search of new nanoscale tools for diverse applications that include (bio)sensing, labeling, targeted imaging, cellular delivery, diagnostics, therapeutics, theranostics, bioelectronics, and biocomputing to name just a few amongst many others. Here, we review this vibrant and growing research area from the perspective of the materials themselves and their unique capabilities. Inorganic nanocrystals such as quantum dots or those made from gold or other (noble) metals along with metal oxides and carbon allotropes are desired as participants in these hybrid materials since they can provide distinctive optical, physical, magnetic, and electrochemical properties. Beyond this, synthetic polymer-based and proteinaceous or viral nanoparticulate materials are also useful in the same role since they can provide a predefined and biocompatible cargo-carrying and targeting capability. The DNA component typically provides sequence-based addressability for probes along with, more recently, unique architectural properties that directly originate from the burgeoning structural DNA field. Additionally, DNA aptamers can also provide specific recognition capabilities against many diverse non-nucleic acid targets across a range of size scales from ions to full protein and cells. In addition to appending DNA to inorganic or polymeric nanoparticles, purely DNA-based nanoparticles have recently surfaced as an excellent assembly platform and have started finding application in areas like sensing, imaging and immunotherapy. We focus on selected and representative nanoparticle-DNA materials and highlight their

  4. COMBINED DETECTION OF CYCLIN D1, P27 AND DNA CONTENT IN ESOPHAGEAL CANCER

    Institute of Scientific and Technical Information of China (English)

    MA Ping; YIN Yuan-qin; WANG Xiao-hua

    2006-01-01

    Objective: To investigate the expressions of cyclin D1 and p27 and DNA content in esophageal cancer and adjacent normal tissues, and to discuss the relationship between them. Methods: The cyclinD1 and p27 were detected by immunohistochemical staining; DNA content was measured by flow cytometry. Results: The positive expression rates of cyclinD1 and p27 in cancer were 45.8% and 33.3% respectively, the DNA content in the positive group of cyclinD1 was higher than that in the negative group of cyclinD1(1.54(0.21 versus 1.08(0.43, P<0.05), while the DNA content and SPF (S-phase fraction) in the positive group of p27 were lower than those in the negative group (1.10(0.19 and 5.56%(5.18% versus 1.66(0.28 and 19.78%(6.12%, P<0.05). Conclusion: The data show that the expression of cyclinD1 and p27 are related to the ontogenesis and progression of esophageal cancer. The combined detection of cyclinD1, p27 and DNA content may be indicators of diagnosis and assessment of esophageal cancer.

  5. Augmentation of French grunt diet description using combined visual and DNA-based analyses

    Science.gov (United States)

    Hargrove, John S.; Parkyn, Daryl C.; Murie, Debra J.; Demopoulos, Amanda W.J.; Austin, James D.

    2012-01-01

    Trophic linkages within a coral-reef ecosystem may be difficult to discern in fish species that reside on, but do not forage on, coral reefs. Furthermore, dietary analysis of fish can be difficult in situations where prey is thoroughly macerated, resulting in many visually unrecognisable food items. The present study examined whether the inclusion of a DNA-based method could improve the identification of prey consumed by French grunt, Haemulon flavolineatum, a reef fish that possesses pharyngeal teeth and forages on soft-bodied prey items. Visual analysis indicated that crustaceans were most abundant numerically (38.9%), followed by sipunculans (31.0%) and polychaete worms (5.2%), with a substantial number of unidentified prey (12.7%). For the subset of prey with both visual and molecular data, there was a marked reduction in the number of unidentified sipunculans (visual – 31.1%, combined &ndash 4.4%), unidentified crustaceans (visual &ndash 15.6%, combined &ndash 6.7%), and unidentified taxa (visual &ndash 11.1%, combined &ndash 0.0%). Utilising results from both methodologies resulted in an increased number of prey placed at the family level (visual &ndash 6, combined &ndash 33) and species level (visual &ndash 0, combined &ndash 4). Although more costly than visual analysis alone, our study demonstrated the feasibility of DNA-based identification of visually unidentifiable prey in the stomach contents of fish.

  6. Perspectives on the combination of radiotherapy and targeted therapy with DNA repair inhibitors in the treatment of pancreatic cancer.

    Science.gov (United States)

    Yang, Shih-Hung; Kuo, Ting-Chun; Wu, Hsu; Guo, Jhe-Cyuan; Hsu, Chiun; Hsu, Chih-Hung; Tien, Yu-Wen; Yeh, Kun-Huei; Cheng, Ann-Lii; Kuo, Sung-Hsin

    2016-08-28

    Pancreatic cancer is highly lethal. Current research that combines radiation with targeted therapy may dramatically improve prognosis. Cancerous cells are characterized by unstable genomes and activation of DNA repair pathways, which are indicated by increased phosphorylation of numerous factors, including H2AX, ATM, ATR, Chk1, Chk2, DNA-PKcs, Rad51, and Ku70/Ku80 heterodimers. Radiotherapy causes DNA damage. Cancer cells can be made more sensitive to the effects of radiation (radiosensitization) through inhibition of DNA repair pathways. The synergistic effects, of two or more combined non-lethal treatments, led to co-administration of chemotherapy and radiosensitization in BRCA-defective cells and patients, with promising results. ATM/Chk2 and ATR/Chk1 pathways are principal regulators of cell cycle arrest, following DNA double-strand or single-strand breaks. DNA double-stranded breaks activate DNA-dependent protein kinase, catalytic subunit (DNA-PKcs). It forms a holoenzyme with Ku70/Ku80 heterodimers, called DNA-PK, which catalyzes the joining of nonhomologous ends. This is the primary repair pathway utilized in human cells after exposure to ionizing radiation. Radiosensitization, induced by inhibitors of ATM, ATR, Chk1, Chk2, Wee1, PP2A, or DNA-PK, has been demonstrated in preclinical pancreatic cancer studies. Clinical trials are underway. Development of agents that inhibit DNA repair pathways to be clinically used in combination with radiotherapy is warranted for the treatment of pancreatic cancer. PMID:27621574

  7. Designing and constructing an 100 bp DNA Ladder by combining PCR and enzyme digestion methods

    Directory of Open Access Journals (Sweden)

    Saidijam M

    2011-05-01

    Full Text Available "n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Molecular DNA markers are one of the most important tools in molecular biology labs. The size of DNA molecules is determined by comparing them with known bands of markers during gel electrophoresis. There are many different protocols to produce these kinds of molecular markers. In this study we have suggested an efficient strategy to produce molecular weight markers in industrial proportions."n"nMethods : To achieve the desired sizes of DNA fragments, a combination of two previously known methods, restriction enzyme digestion and polymerase chain reaction (PCR, were used. The enzymatic digestion process was based on designing and constructing plasmids which equaled in size with the desired length of DNA fragments and produced the desired DNA fragment upon linearization. In the PCR method, the desired length of DNA fragments were cloned in multiple cloning sites of pTZ57R plasmid and in a PCR reaction, the new constructed plasmid was used as a template to produce the final fragment."n"nResults : Upon application of this strategy, 2000 and 3000 bp DNA fragments were produced by enzymatic digestion of plasmids of the same size. Moreover, 100 to 1500 bp fragments were produced during PCR using only a set of

  8. Combination chemotherapy of cancer using the inhibitor of DNA methylation 5-aza-2'-deoxycytidine (decitabine

    Directory of Open Access Journals (Sweden)

    Stephan L

    2015-06-01

    Full Text Available The epigenetic alterations marked by DNA methylation contribute to the malignant transformation of cells by silencing critical genes responsible for the regulation of growth. The potent DNA methylation inhibitor 5-aza-2’-deoxycytidine (decitabine; DAC has shown effectiveness in patients with myeloid malignancies. However, the responses are of short duration. The effectiveness of the DAC therapy may be limited by its incapacity to reactivate enough tumor suppressor genes. Other epigenetic mechanisms, such as the histone modification of target genes, may also hinder gene reactivation by DAC. The dose limiting toxicity of DAC is myelosuppression, which limits the duration of this therapy for clinical use. The clinical effectiveness of DAC may be enhanced by its use in combination with other agents that have diverse mechanisms of action. In this literature review, we summarize the results of preclinical and recent clinical trials of DAC used in combination with other agents to treat cancer. This review was conducted by searching online databases to analyze the available evidence regarding this area of interest. We looked at the combination of DAC with other epigenetic agents, cytotoxic agents, tyrosine kinase inhibitors, biochemical modulators and non-toxic agents. The data compiled suggests that combination epigenetic therapy is feasible, moderately toxic and has promising clinical potential. Preclinical studies showed that some combinations of DAC have additive to synergistic antineoplastic action as compared to DAC alone. The data indicate that combination chemotherapy with DAC merits further investigation. This review may be helpful for the future design of clinical trials using DAC in combination for cancer therapy.

  9. Combining allele frequency uncertainty and population substructure corrections in forensic DNA calculations.

    Science.gov (United States)

    Cowell, Robert

    2016-07-01

    In forensic DNA calculations of relatedness of individuals and in DNA mixture analyses, at least two sources of uncertainty are present concerning the allele frequencies used for evaluating genotype probabilities when evaluating likelihoods. They are: (i) imprecision in the estimates of the allele frequencies in the population by using an inevitably finite database of DNA profiles to estimate them; and (ii) the existence of population substructure. Green and Mortera [6] showed that these effects may be taken into account individually using a common Dirichlet model within a Bayesian network formulation, but that when taken in combination this is not the case; however they suggested an approximation that could be used. Here we develop a slightly different approximation that is shown to be exact in the case of a single individual. We demonstrate the numerical closeness of the approximation using a published database of allele counts, and illustrate the effect of incorporating the approximation into calculations of a recently published statistical model of DNA mixtures. PMID:27231804

  10. Immunogenicity and protective efficacy study using combination of four tuberculosis DNA vaccines

    Institute of Scientific and Technical Information of China (English)

    蔡宏; 田霞; 呼西旦; 潘怡; 李国利; 庄玉辉; 朱玉贤

    2003-01-01

    Immune response and protective efficacy for the combination of four tuberculosis DNA vaccines were evaluated in this study. We obtained 1:200 antibody titers against Ag85B 21d after mice were vaccinated for the first time by four recombinant eukaryotic expression vectors containing coding sequences for Ag85B, MPT-64, MPT-63 and ESAT-6. The titers of Ag85B were elevated to 1:102400 after the second injection and decreased to 1:12800 after the third injection. Antibody titers for MPT-64 and MPT-63 reached 1:25600 21 d after the first vaccination, and were then decreased following the second and third injections. No antigen-specific antibody titer against ESAT-6 was detected in sera harvested from immunized mice at any time. These DNA vaccines evoked specific IFN-λ responses in the spleens of vaccinated mice as well. When challenged with M. tuberculosis H37Rv, we found that the lungs of the vaccinated mice produced 99.8% less bacterial counts than that of the empty-vector control group and the bacterial counts were also significantly less than that of the BCG group. Histopathological analyses showed that the lungs of vaccinated mice produced no obvious caseation while over 50%-70% of the pulmonary parenchyma tissue produced central caseation in the vector control group. Our results indicated that the combination of four tuberculosis DNA vaccines may generate high levels of immune responses and result in better animal protection.

  11. Peptidomics combined with cDNA library unravel the diversity of centipede venom

    DEFF Research Database (Denmark)

    Rong, Mingqiang; Yang, Shilong; Wen, Bo;

    2015-01-01

    showed a larger different to known peptide toxins such spider or scorpion toxins. Moreover, only 29 peptides from this centipede venom were identified with known function. It suggested that our work not only important to understand the composition of centipede venom, but also provide many valuable......UNLABELLED: Centipedes are one of the oldest venomous arthropods using toxin as their weapon to capture prey. But little attention was focused on them and only few centipede toxins were demonstrated with activity on ion channels. Therefore, more deep works are needed to understand the diversity...... of centipede venom. In the present study, we use peptidomics combined with cDNA library to uncover the diversity of centipede Scolopendra subspinipes mutilans L. Koch. 192 peptides were identified by LC-MS/MS and 79 precursors were deduced by cDNA library. Surprisingly, the signal peptides of centipede toxins...

  12. GANN: Genetic algorithm neural networks for the detection of conserved combinations of features in DNA

    Directory of Open Access Journals (Sweden)

    Beiko Robert G

    2005-02-01

    Full Text Available Abstract Background The multitude of motif detection algorithms developed to date have largely focused on the detection of patterns in primary sequence. Since sequence-dependent DNA structure and flexibility may also play a role in protein-DNA interactions, the simultaneous exploration of sequence- and structure-based hypotheses about the composition of binding sites and the ordering of features in a regulatory region should be considered as well. The consideration of structural features requires the development of new detection tools that can deal with data types other than primary sequence. Results GANN (available at http://bioinformatics.org.au/gann is a machine learning tool for the detection of conserved features in DNA. The software suite contains programs to extract different regions of genomic DNA from flat files and convert these sequences to indices that reflect sequence and structural composition or the presence of specific protein binding sites. The machine learning component allows the classification of different types of sequences based on subsamples of these indices, and can identify the best combinations of indices and machine learning architecture for sequence discrimination. Another key feature of GANN is the replicated splitting of data into training and test sets, and the implementation of negative controls. In validation experiments, GANN successfully merged important sequence and structural features to yield good predictive models for synthetic and real regulatory regions. Conclusion GANN is a flexible tool that can search through large sets of sequence and structural feature combinations to identify those that best characterize a set of sequences.

  13. DISSOLVED FREE AMINO ACIDS, COMBINED AMINO ACIDS, AND DNA AS SOURCES OF CARBON AND NITROGEN TO MARINE BACTERIA

    Science.gov (United States)

    Utilization of naturally-occurring dissolved free and combined mino cids (DFAA and DCAA) and dissolved DNA FD-DNA) was studied in batch cultures of bacteria from 2 shallow marine environments. anta Rosa Sound (SRS), Florida, USA, and Flax Pond (FP), Long Island, New York, USA. n ...

  14. Clinical variability in neurohepatic syndrome due to combined mitochondrial DNA depletion and Gaucher disease

    Directory of Open Access Journals (Sweden)

    Julie Harvengt

    2014-01-01

    Full Text Available A 1-year-old girl born to consanguineous parents presented with unexplained liver failure, leading to transplantation at 19 months. Subsequent partial splenectomy for persistent cytopenia showed the presence of foamy cells, and Gaucher disease was confirmed by homozygosity for the p.Leu483Pro mutation in the GBA gene. She was treated by enzyme replacement therapy (ERT. Clinical follow-up showed mild developmental delay, strabismus, nystagmus and oculomotor apraxia. Biochemical studies revealed multiple respiratory chain deficiencies and a mosaic pattern of deficient complex IV immunostaining in liver and fibroblast. Molecular analysis identified a mtDNA depletion syndrome due to the homozygous p.Pro98Leu mutation in MPV17. A younger sister unaffected by mtDNA depletion, presented with pancytopenia and hepatosplenomegaly. ERT for Gaucher disease resulted in visceral normalization without any neurological symptom. A third sister, affected by both conditions, had marked developmental delay, strabismus and ophthalmoplegia but no liver cirrhosis. In conclusion, intrafamilal variability occurs in MPV17-related disease. The combined pathological effect of Gaucher and mitochondrial diseases can negatively impact neurological and liver functions and influence the outcome in consanguineous families. The immunocytochemical staining of OXPHOS protein in tissues and cultured cells is a powerful tool revealing mosaic pattern of deficiency pointing to mtDNA-related mitochondrial disorders.

  15. Combined sequencing of mRNA and DNA from human embryonic stem cells.

    Science.gov (United States)

    Mertes, Florian; Kuhl, Heiner; Wruck, Wasco; Lehrach, Hans; Adjaye, James

    2016-06-01

    Combined transcriptome and whole genome sequencing of the same ultra-low input sample down to single cells is a rapidly evolving approach for the analysis of rare cells. Besides stem cells, rare cells originating from tissues like tumor or biopsies, circulating tumor cells and cells from early embryonic development are under investigation. Herein we describe a universal method applicable for the analysis of minute amounts of sample material (150 to 200 cells) derived from sub-colony structures from human embryonic stem cells. The protocol comprises the combined isolation and separate amplification of poly(A) mRNA and whole genome DNA followed by next generation sequencing. Here we present a detailed description of the method developed and an overview of the results obtained for RNA and whole genome sequencing of human embryonic stem cells, sequencing data is available in the Gene Expression Omnibus (GEO) database under accession number GSE69471.

  16. Determination of DNA adducts by combining acid-catalyzed hydrolysis and chromatographic analysis of the carcinogen-modified nucleobases.

    Science.gov (United States)

    Leung, Elvis M K; Deng, Kailin; Wong, Tin-Yan; Chan, Wan

    2016-01-01

    The commonly used method of analyzing carcinogen-induced DNA adducts involves the hydrolysis of carcinogen-modified DNA samples by using a mixture of enzymes, followed by (32)P-postlabeling or liquid chromatography (LC)-based analyses of carcinogen-modified mononucleotides/nucleosides. In the present study, we report the development and application of a new approach to DNA adduct analysis by combining the H(+)/heat-catalyzed release of carcinogen-modified nucleobases and the use of LC-based methods to analyze DNA adducts. Results showed that heating the carcinogen-modified DNA samples at 70 °C for an extended period of 4 to 6 h in the presence of 0.05% HCl can efficiently induce DNA depurination, releasing the intact carcinogen-modified nucleobases for LC analyses. After optimizing the hydrolysis conditions, DNA samples with C8- and N (2) -modified 2'-deoxyguanosine, as well as N (6) -modified 2'-deoxyadenosine, were synthesized by reacting DNA with 1-nitropyrene, acetaldehyde, and aristolochic acids, respectively. These samples were then hydrolyzed, and the released nucleobase adducts were analyzed using LC-based analytical methods. Analysis results demonstrated a dose-dependent release of target DNA adducts from carcinogen-modified DNA samples, indicating that the developed H(+)/heat-catalyzed hydrolysis method was quantitative. Comparative studies with enzymatic digestion method on carcinogen-modified DNA samples revealed that the two hydrolysis methods did not yield systematically different results.

  17. THE CLONING OF HRNT-1 USING A COMBINATION OF cDNA LIBRARY SCREENING WITH BIOTIN-LABELED PROBE AND RAPID AMPLIFICATION OF cDNA ENDS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To clone the human counterpart of rat ZA73, EST cloned from rat tracheal epithelial (RTE) neoplastic transformed cell model induced by (a-particles radiation by using mRNA differential display. Methods: According to the sequence of rat ZA73, a probe was biotin-labeled to screen human cDNA library, and then the gene sequence was extended by RACE (rapid amplification of cDNA ends). Result: Human gene HRNT-1 (GenBank Accession Number: AF223393) is 4.256 kb in length, with an ORF located in the region between 254 and 3013 bp. 5' UTS (untranslated sequences) is 253 bp, 3' UTS is 1243 bp. Conclusion: The combination of cDNA library screening with biotin-labeled probes and RACE is an effective method to clone full-length cDNA, especially for sequences longer than 2 kb.

  18. MOLECULAR SYSTEMATICS OF IRIDACEAE: A COMBINED ANALYSIS OF FOUR PLASTID DNA SEQUENCE MATRICES

    Directory of Open Access Journals (Sweden)

    M.W. CHASE

    2000-01-01

    Full Text Available Iridaceae are one of the largest families of Lilianae and probably also among the best studied families of monocotyledons. To further evaluate generic, tribal and subfamilial relationships, we have produced four plastid DNA data sets for 57 genera of Iridaceae plus outgroups: rps4, rbcL (both protein coding genes, and the trnL intron snd the trnL-F inter-gene spacer. All four matrices produce highly congruent, although not identical trees, and we thus analysed them in a combined analysis, which produced a highly resolved and well supported topology. In each of the individual trees, some genera or groups of genera are misplaced relative to Goldblatt’s and Rudall’s morphological cladistic studies, but the combined analysis produced a pattern much more similar to these previous ideas of relationships. In the combined tree, all subfamilies were resolved as monophyletic clades, except Nivenioideae, which formed a grade in which Ixioideae were embedded. The achlorophyllous Geosiris (sometimes referred to Geosiridaceae or Burmanniaceae fell within the nivenioid grade. Most of the tribes are monophyletic, except for Ixieae, Watsonieae and Sisyrinchieae, but the topology within Ixioideae is not strongly supported due to extremely low levels of sequence divergence. Isophysis is sister to the rest of the family, and Diplarrhena falls in a well supported position as sister to Irideae/Sisyrinchieae/Tigridieae/Mariceae; Bobartia of Sisyrinchieae is supported as a member of Irideae.

  19. DNA damage and radiosensitizers: M. luteus sensitive sites for misonidazole-TAN combination

    International Nuclear Information System (INIS)

    It has been shown previously that TAN does not enhance production of single-strand breaks (SSB) in DNA of Chinese hamster cells irradiated under hypoxia. In contrast, misonidazole does enhance the yield of SSB, but this SSB enhancement by misonidazole is reduced if TAN is present with misonidazole during irradiation. It was also shown that each of these sensitizers enhances base/sugar damage, measured with the aid of bacterial enzymes (MLS). The results presented here for MLS damage in mammalian cells irradiated in the presence of the misonidazole-TAN combination indicate that the two drugs act independently in enhancing MLS damage, and hence they interact with different types of lesions to produce base/sugar damage. It is proposed that the protection by TAN in mammalian cells observed at the survival level is due to repair of some of that type of damage which would otherwise become a misonidazole-enhanced SSB

  20. Combined effect of radiation and environmental contaminants on DNA repair mechanisms

    International Nuclear Information System (INIS)

    Investigations on the influence of various environmental contamination agents on DNA repair (in combination with irradiation) were reviewed. The agents tested were: detergents (Tween 80, Nonidel P40, Cremophor), aflatoxin B1, furocumarines, drugs (indometacin, cyclophosphamide, vincristine, vinblastine, procarbacine), fluorides, irradiated food constituents, food additives (saccharin), metal ions (Cd, Hg), pesticides (2,4,5-trichlorophenoxyethanol) and infective agents (mycoplasmas). Most of the tests were carried out in vitro with γ-irradiated mouse spleen cells. The detergents and aflatoxin were tested also on E. coli, and irradiated glucose solutions were tested in vivo on Swiss albino mice injected with Salmonella typhimurium TA 1530. Most of the tested agents showed some kind of inhibitory or mutagenic effect. The experiments and results are explained briefly with references to earlier investigations

  1. Analysis of mitochondrial DNA sequences in patients with isolated or combined oxidative phosphorylation system deficiency.

    NARCIS (Netherlands)

    Hinttala, R.; Smeets, R.; Moilanen, J.S.; Ugalde, C.; Uusimaa, J.; Smeitink, J.A.M.; Majamaa, K.

    2006-01-01

    BACKGROUND: Enzyme deficiencies of the oxidative phosphorylation (OXPHOS) system may be caused by mutations in the mitochondrial DNA (mtDNA) or in the nuclear DNA. OBJECTIVE: To analyse the sequences of the mtDNA coding region in 25 patients with OXPHOS system deficiency to identify the underlying g

  2. Determination of chromium combined with DNA, RNA and proteins in chromium-rich brewer's yeast by NAA

    International Nuclear Information System (INIS)

    The content of chromium in the DNA, RNA and protein fractions separated from chromium-rich and normal brewer's yeast was determined by neutron activation analysis (NAA). Our results show that the extracted relative amounts and concentrations of DNA, RNA and proteins have no significant difference for two types of yeast, but the chromium content in DNA, RNA and proteins fractions extracted from the chromium-rich yeast are substantially higher than those from the normal. In addition, the concentration of chromium in DNA is much higher than that in RNA and proteins. It is evident that the inorganic chromium compounds can enter the yeast cell during the yeast cultivation in the chromium-containing culture medium and are converted into organic chromium species, which are combined with DNA, RNA and proteins. (author)

  3. Ligand binding mode to duplex and triplex DNA assessed by combining electrospray tandem mass spectrometry and molecular modeling

    OpenAIRE

    Rosu, Frédéric; Nguyen, Chi-Hung; De Pauw, Edwin; Gabelica, Valérie

    2007-01-01

    In this paper, we report the analysis of seven benzopyridoindole and benzopyridoquinoxaline drugs binding to different duplex DNA and triple helical DNA, using an approach combining electrospray ionization mass spectrometry (ESI-MS), tandem mass spectrometry (MS/MS), and molecular modeling. The ligands were ranked according to the collision energy (CE(50)) necessary to dissociate 50% of the complex with the duplex or the triplex in tandem MS. To determine the probable ligand binding site and ...

  4. Structural fluctuations and quantum transport through DNA molecular wires: a combined molecular dynamics and model Hamiltonian approach

    OpenAIRE

    Gutierrez, R.; Caetano, R.; Woiczikowski, P. B.; Kubar, T.; Elstner, M; Cuniberti, G.

    2009-01-01

    Charge transport through a short DNA oligomer (Dickerson dodecamer) in presence of structural fluctuations is investigated using a hybrid computational methodology based on a combination of quantum mechanical electronic structure calculations and classical molecular dynamics simulations with a model Hamiltonian approach. Based on a fragment orbital description, the DNA electronic structure can be coarse-grained in a very efficient way. The influence of dynamical fluctuations arising either fr...

  5. Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA

    Directory of Open Access Journals (Sweden)

    Metsis Madis

    2008-06-01

    Full Text Available Abstract Background In a traditional electrophoresis mobility shift assay (EMSA a 32P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE in nondenaturing conditions. An extension of this method uses the large population of fragments derived from long genomic regions (approximately 600 kb for the identification of fragments containing protein binding regions. With this method, genomic DNA is fragmented by restriction enzymes, fragments are amplified by PCR, radiolabeled, incubated with nuclear proteins and the resulting DNA-protein complexes are separated by two-dimensional PAGE. Shifted DNA fragments containing protein binding sites are identified by using additional procedures, i. e. gel elution, PCR amplification, cloning and sequencing. Although the method allows simultaneous analysis of a large population of fragments, it is relatively laborious and can be used to detect only high affinity protein binding sites. Here we propose an alternative and straightforward strategy which is based on a combination of native and denaturing PAGE. This strategy allows the identification of DNA fragments containing low as well as high affinity protein binding regions, derived from genomic DNA ( Results We have combined an EMSA-based selection step with subsequent denaturing PAGE for the localization of protein binding regions in long (up to10 kb fragments of genomic DNA. Our strategy consists of the following steps: digestion of genomic DNA with a 4-cutter restriction enzyme (AluI, BsuRI, TruI, etc, separation of low and high molecular weight fractions of resultant DNA fragments, 32P-labeling with Klenow polymerase, traditional EMSA, gel elution and identification of the shifted bands (or smear by denaturing PAGE. The identification of DNA fragments containing protein binding sites is carried out by running the gel-eluted fragments alongside

  6. Exogenous DNA internalisation by sperm cells is improved by combining lipofection and restriction enzyme mediated integration.

    Science.gov (United States)

    Churchil, R R; Gupta, J; Singh, A; Sharma, D

    2011-06-01

    1. Three types of exogenous DNA inserts, i.e. complete linearised pVIVO2-GFP/LacZ vector (9620 bp), the LacZ gene (5317 bp) and the GFP gene (2152 bp) were used to transfect chicken spermatozoa through simple incubation of sperm cells with insert. 2. PCR assay, Dot Blot hybridisation and Southern hybridisation showed the successful internalisation of exogenous DNA by chicken sperm cells. 3. Lipofection and Restriction Enzyme Mediated Integration (REMI) were used to improve the rate of internalisation of exogenous DNA by sperm cells. 4. Results from dot blot as well as Southern hybridisation were semi-quantified and improved exogenous DNA uptake by sperm cells through lipofection and REMI. Stronger signals were observed from hybridisation of LacZ as well as GFP specific probe with the DNA from lipofected exogenous DNA transfected sperm DNA in comparison with those transfected with nude exogenous DNA.

  7. Immune response induced by HPV18 E2 DNA vaccine combined with IL-12

    Institute of Scientific and Technical Information of China (English)

    Yang Fan; SuFang Chen; Zhang Qin; CuiMing Zhu; Chen Jie; YanPing Wan

    2012-01-01

    Objective:To study the effectiveness of human papillomavirus type 18 E2 gene (HPV18-E2) vaccine and interleukin-12(IL-12) on their immune effects. Methods:Thirty-five Balb/c female mice aged 6-8-week-old were randomly divided into five groups (each n =7).The mice were given instramuscular injection of DNA vaccines at 2-week four 4 times(100mg/time).The tails were cut to draw blood at the 0,2,4,6 weeks,and then the mice were executed by eyeball blood letting at 8 weeks.ELISA was used for the quantitative detection of the specific lgG antibody in the srea of Balb/c mice and the cytokine IFN-γ and IL-4 in mice MTT assay.Results: after 8 weeks immunization, antibody IgA in E2 vaccine group andE2 +IL-12 vaccine group was statistically increased as compared with that of control group, and the difference was statistically significant (P<0.01). IFN-γand IL-4 levels of mice spleen lymphocyte culture supernatant inE2 group and E2 + IL-12 were significantly higher than other control groups (P<0.01). the spleen lymphopoiesis activeness of E2 + IL-12 group was significant enhanced as compared with the E2 group, there were statistically significant differences (P<0.01) . Conclusions: E2 DNA vaccine combined with IL-12 immune mice can effectively induce cell-mediated immunity and humoral immune responses, their capabilities of inducing immune response might be more stronger than that of single one.

  8. Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons

    Directory of Open Access Journals (Sweden)

    De Paepe Anne

    2004-02-01

    Full Text Available Abstract Background Activation of proto-oncogenes by DNA amplification is an important mechanism in the development and maintenance of cancer cells. Until recently, identification of the targeted genes relied on labour intensive and time consuming positional cloning methods. In this study, we outline a straightforward and efficient strategy for fast and comprehensive cloning of amplified and overexpressed genes. Results As a proof of principle, we analyzed neuroblastoma cell line IMR-32, with at least two amplification sites along the short arm of chromosome 2. In a first step, overexpressed cDNA clones were isolated using a PCR based subtractive cloning method. Subsequent deposition of these clones on a custom microarray and hybridization with IMR-32 DNA, resulted in the identification of clones that were overexpressed due to gene amplification. Using this approach, amplification of all previously reported amplified genes in this cell line was detected. Furthermore, four additional clones were found to be amplified, including the TEM8 gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences. Conclusions The combinatorial strategy of subtractive cDNA cloning and array CGH analysis allows comprehensive amplicon dissection, which opens perspectives for improved identification of hitherto unknown targeted oncogenes in cancer cells.

  9. CD40-CD40L Interaction in Immunity Against Protozoan Infections

    OpenAIRE

    Mustapha Chamekh

    2007-01-01

    Activation of the immune system against protozoan infections relies particularly on two specific signals provided by cognate interaction of T cells with antigen presenting cells (APCs). The first signal is attributed to binding of the T-cell receptor (TCR) to peptide/MHC complexes on the surface of APCs, whereas the second signal is triggered through binding of several costimulatory molecules on the surface of APCs with their corresponding receptors on T cells. Among these c...

  10. Highly sensitive detection of human IgG using a novel bio-barcode assay combined with DNA chip technology

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhenbao [Central South University, School of Pharmaceutical Sciences (China); Zhou, Bo, E-mail: zhoubo1771@163.com [The Affiliated Zhongda Hospital of Southeast University, Department of Gerontology (China); Wang, Haiqing; Lu, Feng; Liu, Tianjun; Song, Cunxian; Leng, Xigang, E-mail: lengxigyky@163.com [Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College (China)

    2013-09-15

    A simple and ultrasensitive detection of human IgG based on signal amplification using a novel bio-barcode assay and DNA chip technology was developed. The sensing platform was a sandwich system made up of antibody-modified magnetic microparticles (Ab-MMPs)/human IgG/Cy3-labeled single-stranded DNA and antibody-modified gold nanoparticles (Cy3-ssDNA-Ab-AuNPs). The MMPs (2.5 {mu}m in diameter) modified with mouse anti-human IgG monoclonal-antibodies could capture human IgG and further be separated and enriched via a magnetic field. The AuNPs (13 nm in diameter) conjugated with goat anti-human IgG polyclonal-antibodies and Cy3-ssDNA could further combine with the human IgG/Ab-MMP complex. The Cy3-ssDNA on AuNPs was then released by TCEP to hybridize with the DNA chip, thus generating a detectable signal by the fluorescence intensity of Cy3. In order to improve detection sensitivity, a three-level cascaded signal amplification was developed: (1) The MMP enrichment as the first-level; (2) Large quantities of Cy3-ssDNA on AuNPs as the second-level; (3) The Cy3-ssDNA conjugate with DNA chip as the third-level. The highly sensitive technique showed an increased response of the fluorescence intensity to the increased concentration of human IgG through a detection range from 1 pg mL{sup -1} to 10 ng mL{sup -1}. This sensing technique could not only improve the detection sensitivity for the low concentration of human IgG but also present a robust and efficient signal amplification model. The detection method has good stability, specificity, and reproducibility and could be applied in the detection of human IgG in the real samples.

  11. Highly sensitive detection of human IgG using a novel bio-barcode assay combined with DNA chip technology

    International Nuclear Information System (INIS)

    A simple and ultrasensitive detection of human IgG based on signal amplification using a novel bio-barcode assay and DNA chip technology was developed. The sensing platform was a sandwich system made up of antibody-modified magnetic microparticles (Ab-MMPs)/human IgG/Cy3-labeled single-stranded DNA and antibody-modified gold nanoparticles (Cy3-ssDNA-Ab-AuNPs). The MMPs (2.5 μm in diameter) modified with mouse anti-human IgG monoclonal-antibodies could capture human IgG and further be separated and enriched via a magnetic field. The AuNPs (13 nm in diameter) conjugated with goat anti-human IgG polyclonal-antibodies and Cy3-ssDNA could further combine with the human IgG/Ab-MMP complex. The Cy3-ssDNA on AuNPs was then released by TCEP to hybridize with the DNA chip, thus generating a detectable signal by the fluorescence intensity of Cy3. In order to improve detection sensitivity, a three-level cascaded signal amplification was developed: (1) The MMP enrichment as the first-level; (2) Large quantities of Cy3-ssDNA on AuNPs as the second-level; (3) The Cy3-ssDNA conjugate with DNA chip as the third-level. The highly sensitive technique showed an increased response of the fluorescence intensity to the increased concentration of human IgG through a detection range from 1 pg mL−1 to 10 ng mL−1. This sensing technique could not only improve the detection sensitivity for the low concentration of human IgG but also present a robust and efficient signal amplification model. The detection method has good stability, specificity, and reproducibility and could be applied in the detection of human IgG in the real samples

  12. Combined Flow Cytometric Measurement of Two Cell-Surface Antigens and DNA-RNA Content

    OpenAIRE

    sprotocols

    2014-01-01

    Author: Ingrid Schmid Corresponding author ([]()) ### INTRODUCTION Flow cytometry is frequently used to assess nucleic acid content in individual cells. Based on DNA content alone, however, cells in the quiescent G0 phase cannot be discriminated from cells in the proliferative G1 phase, as DNA content remains constant until S-phase entry. In contrast, by measuring RNA content in addition to DNA content, cells can be assigned to G0 and c...

  13. WEE1 kinase targeting combined with DNA-damaging cancer therapy catalyzes mitotic catastrophe

    NARCIS (Netherlands)

    P.C. De Witt Hamer; S.E. Mir; D. Noske; C.J.F. van Noorden; T. Würdinger

    2011-01-01

    WEE1 kinase is a key molecule in maintaining G₂-cell-cycle checkpoint arrest for premitotic DNA repair. Whereas normal cells repair damaged DNA during G₁-arrest, cancer cells often have a deficient G₁-arrest and largely depend on G₂-arrest. The molecular switch for the G₂-M transition is held by WEE

  14. Biochips for cell biology by combined dip-pen nanolithography and DNA-directed protein immobilization.

    Science.gov (United States)

    Arrabito, Giuseppe; Reisewitz, Stephanie; Dehmelt, Leif; Bastiaens, Philippe I; Pignataro, Bruno; Schroeder, Hendrik; Niemeyer, Christof M

    2013-12-20

    A general methodology for patterning of multiple protein ligands with lateral dimensions below those of single cells is described. It employs dip pen nanolithography (DPN) patterning of DNA oligonucleotides which are then used as capture strands for DNA-directed immobilization (DDI) of oligonucleotide-tagged proteins. This study reports the development and optimization of PEG-based liquid ink, used as carrier for the immobilization of alkylamino-labeled DNA oligomers on chemically activated glass surfaces. The resulting DNA arrays have typical spot sizes of 4-5 μm with a pitch of 12 μm micrometer. It is demonstrated that the arrays can be further functionalized with covalent DNA-streptavidin (DNA-STV) conjugates bearing ligands recognized by cells. To this end, biotinylated epidermal growth factor (EGF) is coupled to the DNA-STV conjugates, the resulting constructs are hybridized with the DNA arrays and the resulting surfaces used for the culturing of MCF-7 (human breast adenocarcinoma) cells. Owing to the lateral diffusion of transmembrane proteins in the cell's plasma membrane, specific recruitment and concentration of EGF receptor can be induced specifically at the sites where the ligands are bound on the solid substrate. This is a clear demonstration that this method is suitable for precise functional manipulations of subcellular areas within living cells.

  15. An economical and combined method for rapid and efficient isolation of fungal DNA.

    Science.gov (United States)

    Lech, T; Syguła-Cholewinska, J; Szostak-Kot, J

    2014-12-18

    DNA isolation is a crucial step of conducting genetic studies in any organism. However, this process is quite difficult when studying fungi because of the need to damage the fungal cell walls of specific structures. In this study, we developed a method for the rapid and efficient isolation of fungal DNA based on simultaneous mechanical and enzymatic cell wall degradation. There are several typical modifications of the standard phenol-chloroform DNA extraction method. This method can be modified to degrade the fungal cell wall. The first step of the presented DNA extraction included manual homogenization in modified lysis buffer. Next, enzymatic digestion using 2 enzymes was conducted, including lyticase and proteinase K. To carefully select the most favorable conditions, we developed an economical, rapid, and reliable method for fungal DNA extraction that ensures both high efficiency and proper purity, which are essential for further analyses.

  16. The design and synthesis of novel heterodinuclear complexes combining a DNA-cleaving agent and a DNA-targeting moiety

    NARCIS (Netherlands)

    Hoog, Paul de

    2008-01-01

    Cancer is a leading cause of death worldwide. Nowadays, the treatment of cancer by chemotherapy can consist of a combination of antitumor drugs. Nevertheless, chemotherapy is accompanied by serious side effects and intrinsic and acquired resistance to the drugs. This thesis describes the design and

  17. Enhancing DNA Vaccine Potency by Combining a Strategy to Prolong Dendritic Cell Life and Intracellular Targeting Strategies with a Strategy to Boost CD4+ T Cells

    OpenAIRE

    Kim, Daejin; Hoory, Talia; Wu, T.-C.; Hung, Chien-Fu

    2007-01-01

    Intradermal administration of DNA vaccines, using a gene gun, represents an effective means of delivering DNA directly into professional antigen-presenting cells (APCs) in the skin and thus allows the application of strategies to modify the properties of APCs to enhance DNA vaccine potency. In the current study, we hypothesized that the potency of human papillomavirus (HPV) type 16 E7 DNA vaccines employing intracellular targeting strategies combined with a strategy to prolong the life of den...

  18. A DNA-based system for selecting and displaying the combined result of two input variables

    DEFF Research Database (Denmark)

    Liu, Huajie; Wang, Jianbang; Song, S;

    2015-01-01

    demonstrate this capability in a DNA-based system that takes two input numbers, represented in DNA strands, and returns the result of their multiplication, writing this as a number in a display. Unlike a conventional calculator, this system operates by selecting the result from a library of solutions rather...... than through logic operations. The multiplicative example demonstrated here illustrates a much more general capability—to generate a unique output for any distinct pair of DNA inputs. The system thereby functions as a lookup table and could be a key component in future, more powerful data...

  19. A strategy of tumor treatment in mice with doxorubicin-cyclophosphamide combination based on dendritic cell activation by human double-stranded DNA preparation

    OpenAIRE

    Alyamkina, Ekaterina A; Nikolin, Valeriy P; Popova, Nelly A.; Dolgova, Evgenia V; Proskurina, Anastasia S; Orishchenko, Konstantin E.; Efremov, Yaroslav R.; Chernykh, Elena R.; Ostanin, Alexandr A.; Sidorov, Sergey V; Ponomarenko, Dmitriy M.; Zagrebelniy, Stanislav N; Bogachev, Sergey S.; Shurdov, Mikhail A

    2010-01-01

    Background Immunization of mice with tumor homogenate after combined treatment with cyclophosphamide (CP) and double-stranded DNA (dsDNA) preparation is effective at inhibition of growth of tumor challenged after the treatment. It was assumed that this inhibition might be due to activation of the antigen-presenting cells. The purpose was to develop improved antitumor strategy using mice. We studied the combined action of cytostatics doxorubicin (Dox) plus CP with subsequent dsDNA preparation ...

  20. Indolicidin targets duplex DNA: structural and mechanistic insight through a combination of spectroscopy and microscopy.

    Science.gov (United States)

    Ghosh, Anirban; Kar, Rajiv Kumar; Jana, Jagannath; Saha, Abhijit; Jana, Batakrishna; Krishnamoorthy, Janarthanan; Kumar, Dinesh; Ghosh, Surajit; Chatterjee, Subhrangsu; Bhunia, Anirban

    2014-09-01

    Indolicidin (IR13), a 13-residue antimicrobial peptide from the cathelicidin family, is known to exhibit a broad spectrum of antimicrobial activity against various microorganisms. This peptide inhibits bacterial DNA synthesis resulting in cell filamentation. However, the precise mechanism remains unclear and requires further investigation. The central PWWP motif of IR13 provides a unique structural element that can wrap around, and thus stabilize, duplex B-type DNA structures. Replacements of the central Trp-Trp pair with Ala-Ala, His-His, or Phe-Phe residues in the PxxP motif significantly affects the ability of the peptide to stabilize duplex DNA. Results of microscopy studies in conjunction with spectroscopic data confirm that the DNA duplex is stabilized by IR13, thereby inhibiting DNA replication and transcription. In this study we provide high-resolution structural information on the interaction between indolicidin and DNA, which will be beneficial for the design of novel therapeutic antibiotics based on peptide scaffolds. PMID:25044630

  1. Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA

    Science.gov (United States)

    Gao, Chao; Liu, Shu-yao; Zhang, Xian; Liu, Ying-kai; Qiao, Cong-de; Liu, Zhao-e.

    2016-03-01

    Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4- [4-(N-methyl)styrene] -benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2.

  2. Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA.

    Science.gov (United States)

    Gao, Chao; Liu, Shu-yao; Zhang, Xian; Liu, Ying-kai; Qiao, Cong-de; Liu, Zhao-e

    2016-03-01

    Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4-[4-(N-methyl)styrene]-benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2.

  3. Combined DNA and lipid analyses of sediments reveal changes in Holocene haptophyte and diatom populations in an Antarctic lake

    Science.gov (United States)

    Coolen, Marco J. L.; Muyzer, Gerard; Rijpstra, W. Irene C.; Schouten, Stefan; Volkman, John K.; Sinninghe Damsté, Jaap S.

    2004-06-01

    Preserved ribosomal DNA of planktonic phototrophic algae was recovered from Holocene anoxic sediments of Ace Lake (Antarctica), and the ancient community members were identified based on comparative sequence analysis. The similar concentration profiles of DNA of haptophytes and their traditional lipid biomarkers (alkenones and alkenoates) revealed that fossil rDNA also served as quantitative biomarkers in this environment. The DNA data clearly revealed the presence of six novel phylotypes related to known alkenone and alkenoate-biosynthesizing haptophytes with Isochrysis galbana UIO 102 as their closest relative. The relative abundance of these phylotypes changed as the lake chemistry, particularly salinity, evolved over time. Changes in the alkenone distributions reflect these population changes rather than a physiological response to salinity by a single haptophyte. Using this novel paleo-ecological approach of combining data from lipid biomarkers and preserved DNA, we showed that the post-glacial development of Ace Lake from freshwater basin to marine inlet and the present-day lacustrine saline system caused major qualitative and quantitative changes in the biodiversity of the planktonic populations over time.

  4. Combined immunity of DNA vector and recombinant vaccinia virus expressing Gag proteins of equine infectious anemia virus

    Institute of Scientific and Technical Information of China (English)

    DAI Chunming; ZHANG Xiaoyan; WANG Shuhui; LIU Ying; DUAN Danli; SHEN Rongxian; SHAO Yiming

    2004-01-01

    In order to develop a new vaccine candidate for equine infectious anemia virus (EIAV), gag gene of Chinese donkey leukocyte attenuated strain (EIAV DLV) and its parental virulent strain (EIAV LN) were inserted respectively into the TK region of the Tiantan strain (VV) of vaccinia virus by homologous recombination and the positive clone was confirmed by blue plaque assay. Protein expression was examined by Western blot. Prime and prime-boost procedures were used to immunize mice with two DNA vectors and two recombinant vaccinia viruses expressing EIAV Gag proteins. The results showed that the specific lysis of CTL responses in the DNA+rVV groups was stronger than those in the DNA groups, amounting to 31%. Although the levels of specific antibodies were not significantly different, we could conclude that the recombinant vaccinia virus could boost the cellular responses following DNA vector priming. There was no detectable difference between the immune responses induced by DLV and LN Gag proteins. This data demonstrates that the combined immunity of DNA vector and recombinant vaccinia virus expressing EIAV gag proteins, utilizing the prime-boost procedure, can drive immunized mice to produce powerful cellular responses. These results lay an important foundation for the development of a new EIAV genetic engineering vaccine.

  5. Simultaneous measurement of DNA motor protein conformation and activity with combined optical trap and single-molecule fluorescence

    Science.gov (United States)

    Chemla, Yann

    2013-03-01

    We present single-molecule measurements of Superfamily 1 UvrD helicase DNA unwinding that reveal directly how helicase stoichiometry and conformation regulate motor activity. Using a new instrument that combines high resolution optical tweezers with single-molecule fluorescence microscopy, we record DNA unwinding activity with base pair-scale resolution (via optical tweezers) simultaneously with helicase stoichiometry and conformation (via fluorescence). Quantifying the fluorescence signal from labeled UvrD, we observe that pairs of UvrD molecules are required for long distance unwinding but that individual molecules exhibit limited, non-processive unwinding activity. UvrD is also known to exhibit two different conformations, `closed' and `open', based on the orientation of its 2B regulatory domain. The function of these conformations has remained elusive. Measuring the fluorescence of FRET labeled proteins, we detect directly the conformation of the 2B domain of individual UvrD molecules during unwinding activity. We observe that UvrD is in the `closed' conformation during DNA unwinding but surprisingly switches to the `open' conformation upon reversal of helicase direction, i.e. when UvrD switches strands and translocates on the opposing strand with the DNA junction rezipping behind it. We hypothesize that the 2B domain acts as a conformational switch that controls DNA unwinding vs. re-annealing. Work supported by NSF (PHY-082261, Center for the Physics of Living Cells) and NIH (R21 RR025341A)

  6. Combination and cleavage of HBV DNA fragments by triple helix-forming oligonucleotides modified with manganese porphyrin in vitro

    Institute of Scientific and Technical Information of China (English)

    光丽霞; 袁发焕; 奚敏; 赵聪敏; 刘立; 温恩懿; 艾友萍

    2003-01-01

    Objective To observe the ability of triple helix-forming oligonucleotides (TFOs) modified with manganese porphyrin to combine with and cleave HBV DNA fractions. Methods TFO were modified with manganese porphyrin and acridines, and then reacted with the 32P labeled HBV DNA fragments at 37℃ in vitro (pH 7.4). Electrophoretic mobility shift assays and Dnase Ⅰ footprinting tests were used to show the affinity and specificity of TFO to bind to target sequences. The ability of TFO to cleave HBV DNA fragments was tested by cleavage experiments. Results TFO modified with manganese porphyrin and acridine could bind to the target sequence in a sequence-dependent manner, with a Kd value of 3.5×10-7 mol/L and a relative affinity of 0.008. In the presence of potassium monopersulfate (KHSO5), TFO modified with manganese porphyrin and acridine could cleave the target sequence where the triplex DNA was formed. Conclusion In the presence of KHSO5, TFO modified with manganese porphyrin and acridine could bind and cleave the target HBV-DNA in a sequence-dependent manner.

  7. Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays

    Directory of Open Access Journals (Sweden)

    Yu Jingjing

    2011-11-01

    Full Text Available Abstract Background The processes that compose expression of a given gene are far more complex than previously thought presenting unprecedented conceptual and mechanistic challenges that require development of new tools. Chromatin structure, which is regulated by DNA methylation and histone modification, is at the center of gene regulation. Immunoprecipitations of chromatin (ChIP and methylated DNA (MeDIP represent a major achievement in this area that allow researchers to probe chromatin modifications as well as specific protein-DNA interactions in vivo and to estimate the density of proteins at specific sites genome-wide. Although a critical component of chromatin structure, DNA methylation has often been studied independently of other chromatin events and transcription. Results To allow simultaneous measurements of DNA methylation with other genomic processes, we developed and validated a simple and easy-to-use high throughput microplate-based platform for analysis of DNA methylation. Compared to the traditional beads-based MeDIP the microplate MeDIP was more sensitive and had lower non-specific binding. We integrated the MeDIP method with a microplate ChIP assay which allows measurements of both DNA methylation and histone marks at the same time, Matrix ChIP-MeDIP platform. We illustrated several applications of this platform to relate DNA methylation, with chromatin and transcription events at selected genes in cultured cells, human cancer and in a model of diabetic kidney disease. Conclusion The high throughput capacity of Matrix ChIP-MeDIP to profile tens and potentially hundreds of different genomic events at the same time as DNA methylation represents a powerful platform to explore complex genomic mechanism at selected genes in cultured cells and in whole tissues. In this regard, Matrix ChIP-MeDIP should be useful to complement genome-wide studies where the rich chromatin and transcription database resources provide fruitful foundation

  8. Combining Electro-Osmotic Flow and FTA® Paper for DNA Analysis on Microfluidic Devices

    Directory of Open Access Journals (Sweden)

    Ryan Wimbles

    2016-07-01

    Full Text Available FTA® paper can be used to protect a variety of biological samples prior to analysis, facilitating ease-of-transport to laboratories or long-term archive storage. The use of FTA® paper as a solid phase eradicates the need to elute the nucleic acids from the matrix prior to DNA amplification, enabling both DNA purification and polymerase chain reaction (PCR-based DNA amplification to be performed in a single chamber on the microfluidic device. A disc of FTA® paper, containing a biological sample, was placed within the microfluidic device on top of wax-encapsulated DNA amplification reagents. The disc containing the biological sample was then cleaned up using Tris-EDTA (TE buffer, which was passed over the disc, via electro-osmotic flow, in order to remove any potential inhibitors of downstream processes. DNA amplification was successfully performed (from buccal cells, whole blood and semen using a Peltier thermal cycling system, whereupon the stored PCR reagents were released during the initial denaturing step due to the wax barrier melting between the FTA® disc and PCR reagents. Such a system offers advantages in terms of a simple sample introduction interface and the ability to process archived samples in an integrated microfluidic environment with minimal risk of contamination.

  9. Detection of Genetically Modified Crops by Combination of Multiplex PCR and Low-density DNA Microarray1

    Institute of Scientific and Technical Information of China (English)

    PING-PING ZHOU; JIAN-ZHONG ZHANG; YUAN-HAI YOU; YONG-NING WU

    2008-01-01

    Objective To develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray.Methods Two sets of the multiplex PCR system were used to amplify the target genes in genetically modified(GM)soybean.Seventeen capture probes(PCR products)and 17 pairs of corresponding primers were designed according to the genetic characteristies of Rroundup Ready soybean(GTS40-3-2),maize (Mort810,Nk603,GA21),canola(T45,MS1/RF1),and rice(SCK)in many identified GM crops.All of the probes were categorized and identified as species-specific probes.One negative probe and one positive control probe were uscd to assess the efficiency of all reactions,and therefore eliminate any false positive and negative results.After multiplex PCR reaction,amplicons were adulterated with Cy5-dUTP and hvbridized with DNA microarray.The array was then scanned to display the specific hybridization signals of target genes.The assay was applied to the analysis of sample of ccrtified transgenic soybean (Roundup Ready GTS40-3-2)and canola(MS1/RF1). Results A combination technique of multiplex PCR and DNA microarray was successfully developed to identify multi-target genes in Roundup Ready soybean and MS1/RF1 canola with a great specificity and reliability.Reliable identification of genetic characteristics of Roundup Ready of GM soybean from genetically modified crops was achieved at 0.5% transgenic events,indicating a high sensitivity. Conclusion A combination technique of multiplex PCR and low-density DNA microarray can reliably detect and identify the genetically modified crops.

  10. Combination of gefitinib and DNA methylation inhibitor decitabine exerts synergistic anti-cancer activity in colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Yun-feng Lou

    Full Text Available Despite recent advances in the treatment of human colon cancer, the chemotherapy efficacy against colon cancer is still unsatisfactory. In the present study, effects of concomitant inhibition of the epidermal growth factor receptor (EGFR and DNA methyltransferase were examined in human colon cancer cells. We demonstrated that decitabine (a DNA methyltransferase inhibitor synergized with gefitinib (an EGFR inhibitor to reduce cell viability and colony formation in SW1116 and LOVO cells. However, the combination of the two compounds displayed minimal toxicity to NCM460 cells, a normal human colon mucosal epithelial cell line. The combination was also more effective at inhibiting the AKT/mTOR/S6 kinase pathway. In addition, the combination of decitabine with gefitinib markedly inhibited colon cancer cell migration. Furthermore, gefitinib synergistically enhanced decitabine-induced cytotoxicity was primarily due to apoptosis as shown by Annexin V labeling that was attenuated by z-VAD-fmk, a pan caspase inhibitor. Concomitantly, cell apoptosis resulting from the co-treatment of gefitinib and decitabine was accompanied by induction of BAX, cleaved caspase 3 and cleaved PARP, along with reduction of Bcl-2 compared to treatment with either drug alone. Interestingly, combined treatment with these two drugs increased the expression of XIAP-associated factor 1 (XAF1 which play an important role in cell apoptosis. Moreover, small interfering RNA (siRNA depletion of XAF1 significantly attenuated colon cancer cells apoptosis induced by the combination of the two drugs. Our findings suggested that gefitinib in combination with decitabine exerted enhanced cell apoptosis in colon cancer cells were involved in mitochondrial-mediated pathway and induction of XAF1 expression. In conclusion, based on the observations from our study, we suggested that the combined administration of these two drugs might be considered as a novel therapeutic regimen for treating colon

  11. Quantitation of DNA methyltransferase activity via chronocoulometry in combination with rolling chain amplification.

    Science.gov (United States)

    Ji, Jingjing; Liu, Yuanjian; Wei, Wei; Zhang, Yuanjian; Liu, Songqin

    2016-11-15

    In this paper, a rolling chain amplification (RCA) strategy was proposed for chronocoulometric detection of DNA methyltransferase (MTase) activity. Briefly, after the double DNA helix structure was assembled on the surface of gold electrode, it was first methylated by M. SssI MTase and then RCA was realized in the presence of E. coli and phi29 DNA polymerase. Successively, numerous hexaammineruthenium (III) chloride ([Ru(NH3)6)(3+), RuHex) were adsorbed on replicons by electrostatic interaction and generated a large electrochemical readout, the signal was "on". On the contrary, in the absence of M. SssI MTase, the methylated CpG site in the unmethylated double DNA helix structure could be specifically recognized and cleaved by HpaII, resulting in a disconnection of RCA from the electrode. This led seldom RuHex to be absorbed onto the surface of electrode, the signal was "off". Based on the proposed strategy, the activity of M. SssI MTase was assayed in the range of 0.5-60U/mL with a detection limit of 0.09U/mL (S/N=3). In addition, the inhibition of procaine and epicatechin on M. SssI MTase activity was evaluated. When the proposed method was applied in complex matrix such as human serum samples, acceptable accuracy, precision and high sensitivity were achieved. Therefore, the proposed method was a potential useful mean for clinical diagnosis and drug development. PMID:27155113

  12. Berberine in combination with cisplatin suppresses breast cancer cell growth through induction of DNA breaks and caspase-3-dependent apoptosis.

    Science.gov (United States)

    Zhao, Yuwan; Jing, Zuolei; Li, Yan; Mao, Weifeng

    2016-07-01

    Berberine (BBR) is an isoquinoline alkaloid extracted from medicinal plants such as Hydrastis canadensis, Berberis aristata and Coptis chinensis. BBR displays a number of beneficial roles in the treatment of various types of cancers, yet the precise mechanisms of its action remain unclear. Cisplatin is an effective cancer chemotherapeutic agent and functions by generating DNA damage, promoting DNA damage-induced cell cycle arrest and apoptosis; however, its efficacy is challenged by the resistance of tumor cells in clinical application. The aim of the present study was to investigate the effects of BBR in combination with cisplatin on human breast cancer cells. MTT assay showed that BBR inhibited breast cancer MCF-7 cell growth with a 50% inhibitory concentration (IC50) value of 52.178±1.593 µM and the IC50 value of cisplatin was 49.541±1.618 µM, while in combination with 26 µM BBR, the IC50 value of cisplatin was 5.759±0.76 µM. BBR sensitized the MCF-7 cells to cisplatin in a time- and dose-dependent manner. After treatment of BBR and cisplatin, the cellular pro-apoptotic capase-3 and cleaved capspase-3 and caspase-9 were upregulated and the anti-apoptotic Bcl-2 was downregulated. Importantly, BBR restrained the expression of cellular PCNA, and immunofluoresence analysis of γH2AX showed that BBR increased the DNA damages induced by cisplatin. Taken together, the results demonstrated that BBR sensitized MCF-7 cells to cisplatin through induction of DNA breaks and caspase-3-dependent apoptosis. PMID:27177238

  13. Construction of two fluorescence-labeled non-combined DNA index system miniSTR multiplex systems to analyze degraded DNA samples in the Chinese Han Population.

    Science.gov (United States)

    Bai, Xue; Li, Shujin; Cong, Bin; Li, Xia; Guo, Xia; He, Lujun; Ye, Jian; Pei, Li

    2010-09-01

    MiniSTR loci have been demonstrated to be an effective approach in recovering genetic information from degraded specimens, because of the reduced PCR amplicon sizes which improved the PCR efficiency. Eight non-combined DNA index system miniSTR loci suitable for the Chinese Han Population were analyzed in 300 unrelated Chinese Han individuals using two novel five fluorescence-labeled miniSTR multiplex systems(multiplex I: D10S1248, D2S441, D1S1677 and D9S2157; multiplex II: D9S1122, D10S1435, D12ATA63, D2S1776 and Amelogenin). The allele frequency distribution and forensic parameters in the Chinese Han Population were reported in this article. The Exact Test demonstrated that all loci surveyed here were found to be no deviation from Hardy-Weinberg equilibrium. The accumulated power of discrimination and power of exclusion for the eight loci were 0.999999992 and 0.98, respectively. The highly degraded DNA from artificially degraded samples and the degraded forensic case work samples was assessed with the two miniSTR multiplex systems, and the results showed that the systems were quite effective.

  14. Combining peptide and DNA for protein assay: CRIP1 detection for breast cancer staging.

    Science.gov (United States)

    Xie, Haona; Li, Hao; Huang, Yue; Wang, Xiaoying; Yin, Yongmei; Li, Genxi

    2014-01-01

    In this work, a novel method for a protein assay is proposed which uses the specific protein-binding peptide of the target protein and sequence-specific DNA to interact with the target as the capture and detection probe, respectively. Meanwhile, since the DNA sequence can be coupled with gold nanoparticles to amplify the signal readout, a sensitive and easily operated method for protein assay is developed. We have also employed a transcription factor named as cysteine-rich intestinal protein 1 (CRIP1), which has been identified as an ideal biomarker for staging of breast cancer, as the model protein for this study. With the proposed method, CRIP1 can be determined in a linear range from 1.25 to 10.13 ng/mL, with a detection limit of 1.25 ng/mL. Furthermore, the proposed method can be directly used to assay CRIP1 in tissue samples. Owing to its desirable sensitivity, excellent reproducibility, and high selectivity, the proposed method may hold great potential in clinical practice in the future.

  15. Effects of combined immune therapy on survival and Th1/rh2 cytokine balance in rat orthotopic liver transplantation

    Institute of Scientific and Technical Information of China (English)

    CAO Hui; LIU Hua; WU Zhi-yong

    2007-01-01

    Background The induction of immune tolerance and suppression of allograft rejection has become the focus in the study of liver transplantation. The effect of immune therapy with anti-CD40L mAb alone or in combination with cyclosporine A (CsA) on the recipient survival and Th1/Th2 cytokine profile was studied to elucidate its immunological mechanism and role in rat orthotopic liver transplantation.Methods The model of rat orthotopic liver transplantation was established by modified Kamada's technique.Recipients were divided into group A (control group): SD→SD; group B (group of rejection): SD→Wistar without any treatment; group C: SD→Wistar with CsA monotherapy from day 1 to day 5; and group D: SD→Wistar with CsA from day 1 to day 5 and anti-CD40L mAb on day 0 and day 2. The survival of the recipients in all groups was observed and ELISA technique was used to detect the level of cytokines in peripheral blood on post-transplant day 7.Results The survival period of recipients in groups A (>60 days) and D (>60 days) was significantly longer than that in group B (13.8±2.4 days). The serum levels of interleukin 2 (IL-2) and interferon y in group B were significantly higher than those in other groups; the level of tumor necrosis factor α was higher but not statistically significant. In contrast, the serum levels of IL-4 and IL-10 in group D were elevated more significantly than those in group B (P<0.05).Conclusions Combined immune therapy can prolong the survival of allografts. Increased expression of Th2 cytokines, which is closely related to the induction of tolerance and suppression of rejection, is beneficial to the long-term survival of recipients and allografts.

  16. Venom landscapes: mining the complexity of spider venoms via a combined cDNA and mass spectrometric approach.

    Science.gov (United States)

    Escoubas, Pierre; Sollod, Brianna; King, Glenn F

    2006-05-01

    The complexity of Australian funnel-web spider venoms has been explored via the combined use of MALDI-TOF mass spectrometry coupled with chromatographic separation and the analysis of venom-gland cDNA libraries. The results show that these venoms are far more complex than previously realized. We show that the venoms of Australian funnel-web spiders contain many hundreds of peptides that follow a bimodal distribution, with about 75% of the peptides having a mass of 3000-5000 Da. The mass spectral data were validated by matching the experimentally observed masses with those predicted from peptide sequences derived from analysis of venom-gland cDNA libraries. We show that multiple isoforms of these peptides are found in small chromatographic windows, which suggests that the wide distribution of close molecular weights among the chromatographic fractions probably reflects a diversity of structures and physicochemical properties. The combination of all predicted and measured parameters permits the interpretation of three-dimensional 'venom landscapes' derived from LC-MALDI analysis. We propose that these venom landscapes might have predictive value for the discovery of various groups of pharmacologically distinct toxins in complex venoms.

  17. Combined DNA, toxicological and heavy metal analyses provides an auditing toolkit to improve pharmacovigilance of traditional Chinese medicine (TCM)

    Science.gov (United States)

    Coghlan, Megan L.; Maker, Garth; Crighton, Elly; Haile, James; Murray, Dáithí C.; White, Nicole E.; Byard, Roger W.; Bellgard, Matthew I.; Mullaney, Ian; Trengove, Robert; Allcock, Richard J. N.; Nash, Christine; Hoban, Claire; Jarrett, Kevin; Edwards, Ross; Musgrave, Ian F.; Bunce, Michael

    2015-12-01

    Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines.

  18. Study on the Detection of Telomerase Activity by Combining DNA Sequence Analysis with TRAP

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Telomeric repeat amplification protocol (TRAP) is now aconventional assay for detecting telomerase activity. However, this method presents problems owing to tedious quantitation, radioisotopic handling. In order to alleviate these inconveniences, a novel telomerase DNA sequencing assay together with TRAP to detect human telomerase activity was developed. It was used to detect telomerase activity in Hela, HLF, MCF, K562, SMMC-7721 cells, Leukocytes and RNase-pretreated or heat-treated cells as control. Telomerase activity assayed by this method was positive when the number of K562 cells examined was 102,103, and 104. The telomerase activity depended on the number of K562 cells used in the assay. Telomerase activity of Rnase-pretreated cells or heat-treated cells, and human normal peripheral blood leukocyte(Leu) were negative. The result of this method was available within a few hours and was handled without radioisotope. Further studies should be taken to detect telomerase activity in quantitation.

  19. The effect of β-elemene combined with irradiation on DNA damage and repair in A549 cells

    International Nuclear Information System (INIS)

    Objective: To study if β-elemene can increase radiation-induced deoxyribonucleic acid (DNA) damage and decrease the damage repair. Methods: Exponentially growing human lung adenocarcinoma cells (A549) were exposed to 10 or 20 μg/ml β-elemene for 24 h before irradiation.The effect of β-elemene on the in vitro radiosensitivity of A549 cells was evaluated using clonogenic assay. DNA damage and repair were evaluated using comet assay. Results: Exposure to β-elemene before irradiation increased the radiosensitivity of A549 cells. The SERD0 for 10 μg/ml and 20 μg/ml β-elemene was 1.55 and 1.64, respectively. The SERDq for 10 μg/ml and 20 μg/ml β-elemene was 1.43 and 1.75, respectively. Combined treatment, comparing to irradiation or β-elemene treatment alone, induced higher levels of DNA damage and slower rate of damage repair. A549 cells exposed to 20 μg/ml β-elemene followed by irradiation showed a higher levels of tail moment (TM) than those exposed to irradiation or β-elemene alone at 0 h, 2 h, 6 h and 24 h after irradiation. The TM of the three groups at 0 h, 2 h, 6 h and 24 h after irradiation was 7.16±2.61, 0.95±0.65 and 1.81±1.23 (F=231.24, P<0.01), 3.65±2.06, 0.11±0.07 and 1.58±1.40(F=90.22, P<0.01), 2.09±0.83, 0.1±0.05 and 0.45±0.25 (F=238.44, P<0.01), 1.45±1.37, 0.11±0.08 and 0.60±0.40 (F=38.94, P<0.01), respectively. Conclusions: β-elemene can enhance the radiosensitivity of A549 cells through the enhancement of DNA damage and the inhibition of DNA damage repair. (authors)

  20. Chromosome damage induced by DNA topoisomerase II inhibitors combined with g-radiation in vitro

    Directory of Open Access Journals (Sweden)

    Maria Cristina P. Araújo

    1998-09-01

    Full Text Available Combined radiation and antineoplastic drug treatment have important applications in cancer therapy. In the present work, an evaluation was made of two known topoisomerase II inhibitors, doxorubicin (DXR and mitoxantrone (MXN, with g-radiation. The effects of DXR or MXN on g-radiation-induced chromosome aberrations in Chinese hamster ovary (CHO cells were analyzed. Two concentrations of each drug, 0.5 and 1.0 µg/ml DXR, and 0.02 and 0.04 µg/ml MXN, were applied in combination with two doses of g-radiation (20 and 40 cGy. A significant potentiating effect on chromosomal aberrations was observed in CHO cells exposed to 1.0 µg/ml DXR plus 40 cGy. In the other tests, the combination of g-radiation with DXR or MXN gave approximately additive effects. Reduced mitotic indices reflected higher toxicity of the drugs when combined with radiation.A associação de radiação ionizante com drogas antineoplásicas tem importante aplicação na terapia do câncer. No presente trabalho, foram avaliados os efeitos de dois inibidores de topoisomerase II, doxorubicina (DXR e mitoxantrona (MXN, sobre as aberrações cromossômicas induzidas pelas radiações-g em células do ovário de hamster chinês (CHO. Foram usadas as concentrações 0,5 e 1,0 mg/ml de DXR e 0,02 e 0,04 mg/ml de MXN, combinadas com duas doses de radiações gama (20 e 40 cGy. Um significativo efeito potenciador das aberrações cromossômicas foi observado em células CHO tratadas com 1,0 mg/ml de DXR e expostas a 40 cGy de radiação. Nos outros testes, a combinação da radiação-g com a DXR ou MXN apresentou um efeito próximo ao aditivo. A redução dos índices mitóticos refletiu a alta citotoxicidade das drogas quando combinadas às radiações-g.

  1. Classification of Breast Cancer Subtypes by combining Gene Expression and DNA Methylation Data

    DEFF Research Database (Denmark)

    List, Markus; Hauschild, Anne-Christin; Tan, Qihua;

    2014-01-01

    expression data for hundreds of patients, the challenge is to extract a minimal optimal set of genes with good prognostic properties from a large bulk of genes making a moderate contribution to classification. Several studies have successfully applied machine learning algorithms to solve this so-called gene...... on the transcriptomic, but also on an epigenetic level. We compared so-called random forest derived classification models based on gene expression and methylation data alone, to a model based on the combined features and to a model based on the gold standard PAM50. We obtained bootstrap errors of 10...

  2. Cations form sequence selective motifs within DNA grooves via a combination of cation-pi and ion-dipole/hydrogen bond interactions.

    Directory of Open Access Journals (Sweden)

    Mikaela Stewart

    Full Text Available The fine conformational subtleties of DNA structure modulate many fundamental cellular processes including gene activation/repression, cellular division, and DNA repair. Most of these cellular processes rely on the conformational heterogeneity of specific DNA sequences. Factors including those structural characteristics inherent in the particular base sequence as well as those induced through interaction with solvent components combine to produce fine DNA structural variation including helical flexibility and conformation. Cation-pi interactions between solvent cations or their first hydration shell waters and the faces of DNA bases form sequence selectively and contribute to DNA structural heterogeneity. In this paper, we detect and characterize the binding patterns found in cation-pi interactions between solvent cations and DNA bases in a set of high resolution x-ray crystal structures. Specifically, we found that monovalent cations (Tl⁺ and the polarized first hydration shell waters of divalent cations (Mg²⁺, Ca²⁺ form cation-pi interactions with DNA bases stabilizing unstacked conformations. When these cation-pi interactions are combined with electrostatic interactions a pattern of specific binding motifs is formed within the grooves.

  3. Combination of small interfering RNAs mediates greater suppression on hepatitis B virus cccDNA in HepG2.2.15 cells

    Institute of Scientific and Technical Information of China (English)

    Xiao-Min Xin; Gui-Qiu U; Ying-Yu Jin; Min Zhuang; Di Li

    2008-01-01

    AIM: To observe the inhibition of hepatitis B virus (HBV) replication and expression in HepG2.2.15 cells by combination of small interfering RNAs (siRNAs).METHODS: Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells.At 48 h,72 h and 96 h after transfection,culture media were collected and cells were harvested for HBV replication assay.HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay (ELISA).Intracellular viral DNA and covalently closed circular DNA (cccDNA)were quantified by real-time polymerase chain reaction (PCR).HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR (RT-PCR).RESULTS: siRNAs showed marked anti-HBV effects.siRNAs could specifically inhibit the expression of HBsAg and the replication of HBV DNA in a dosedependent manner.Furthermore,combination of siRNAs,compared with individual use of each siRNA,exerted a stronger inhibition on antigen expression and viral replication.More importantly,combination of siRNAs significantly suppressed HBV cccDNA amplification.CONCLUSION: Combination of siRNAs mediates a stronger inhibition on viral replication and antigen expression in HepG2.2.15 cells,especially on cccDNA amplification.

  4. Combinations of various CpG motifs cloned into plasmid backbone modulate and enhance protective immunity of viral replicon DNA anthrax vaccines.

    Science.gov (United States)

    Yu, Yun-Zhou; Ma, Yao; Xu, Wen-Hui; Wang, Shuang; Sun, Zhi-Wei

    2015-08-01

    DNA vaccines are generally weak stimulators of the immune system. Fortunately, their efficacy can be improved using a viral replicon vector or by the addition of immunostimulatory CpG motifs, although the design of these engineered DNA vectors requires optimization. Our results clearly suggest that multiple copies of three types of CpG motifs or combinations of various types of CpG motifs cloned into a viral replicon vector backbone with strong immunostimulatory activities on human PBMC are efficient adjuvants for these DNA vaccines to modulate and enhance protective immunity against anthrax, although modifications with these different CpG forms in vivo elicited inconsistent immune response profiles. Modification with more copies of CpG motifs elicited more potent adjuvant effects leading to the generation of enhanced immunity, which indicated a CpG motif dose-dependent enhancement of antigen-specific immune responses. Notably, the enhanced and/or synchronous adjuvant effects were observed in modification with combinations of two different types of CpG motifs, which provides not only a contribution to the knowledge base on the adjuvant activities of CpG motifs combinations but also implications for the rational design of optimal DNA vaccines with combinations of CpG motifs as "built-in" adjuvants. We describe an efficient strategy to design and optimize DNA vaccines by the addition of combined immunostimulatory CpG motifs in a viral replicon DNA plasmid to produce strong immune responses, which indicates that the CpG-modified viral replicon DNA plasmid may be desirable for use as vector of DNA vaccines. PMID:25265876

  5. Combined TLR7/8 and TLR9 ligands potentiate the activity of a Schistosoma japonicum DNA vaccine.

    Directory of Open Access Journals (Sweden)

    Xuefeng Wang

    Full Text Available BACKGROUND: Toll-like receptor (TLR ligands have been explored as vaccine adjuvants for tumor and virus immunotherapy, but few TLR ligands affecting schistosoma vaccines have been characterized. Previously, we developed a partially protective DNA vaccine encoding the 26-kDa glutathione S-transferase of Schistosoma japonicum (pVAX1-Sj26GST. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we evaluated a TLR7/8 ligand (R848 and a TLR9 ligand (CpG oligodeoxynucleotides, or CpG as adjuvants for pVAX1-Sj26GST and assessed their effects on the immune system and protection against S. japonicum. We show that combining CpG and R848 with pVAX1-Sj26GST immunization significantly increases splenocyte proliferation and IgG and IgG2a levels, decreases CD4(+CD25(+Foxp3(+ regulatory T cells (Treg frequency in vivo, and enhances protection against S. japonicum. CpG and R848 inhibited Treg-mediated immunosuppression, upregulated the production of interferon (IFN-γ, tumor necrosis factor (TNF-α, interleukin (IL-4, IL-10, IL-2, and IL-6, and decreased Foxp3 expression in vitro, which may contribute to prevent Treg suppression and conversion during vaccination and allow expansion of antigen-specific T cells against pathogens. CONCLUSIONS: Our data shows that selective TLR ligands can increase the protective efficacy of DNA vaccines against schistosomiasis, potentially through combined antagonism of Treg-mediated immunosuppression and conversion.

  6. Transformation of primary human embryonic kidney cells to anchorage independence by a combination of BK virus DNA and the Harvey-ras oncogene

    International Nuclear Information System (INIS)

    Primary human embryonic kidney (HEK) cells were transformed by a focus assay with BK virus (BKV) DNA molecularly cloned at its unique EcoRI site. Both viral DNA sequences and viral tumor antigens were present and expressed in all the foci that the authors examined. However, cells isolated from foci were incapable of growth in soft agar. They then examined the transformation of HEK cells after their transfection with a combination of BKV DNA and either the normal or the activated form of the human Ha-ras oncogene (EJ c-Ha-ras-1). Only the cells transfected with a combination of BKV DNA and the activated form of Ha-ras DNAs were present in the transformed colonies. BKV tumor antigens and the Ha-ras p21 protein were also expressed

  7. Molecular mechanisms and reasons of radiation antagonism, self-induced radioprotective effect and hormesis at combined action of ionizing radiation and free radicals on DNA

    International Nuclear Information System (INIS)

    The time-dependent dynamics of formation, relaxation and auto-reparation of double breaks of DNA macromolecules at the combine radiation action and non-tradition processes of degradation were considered. The auto-repairing of DNA double breaks is connected with the peculiarities of long-range interaction of nucleotide charges, atoms and molecules in the intracellular milieu. On the base of the analysis and correlation of these processes the time-dependent theory for DNA degradation was created, including hormesis phenomenon, radiation antagonism, the validity of anomaly influence of low and large doses at sharp and chronic radiation and others effects

  8. Study the effects of metallic ions on the combination of DNA and histones with molecular combing technique

    Institute of Scientific and Technical Information of China (English)

    LIU Yuying; WANG Pengye; DOU Shuoxing; XIE Ping; WANG Weichi; YIN Huawei

    2005-01-01

    The effects of monovalent (Na+, K+) and divalent (Mg2+, Ca2+, Mn2+) ions on the interaction between DNA and histone are studied using the molecular combing technique. λ-DNA molecules and DNA-histone complexes incubated with metal cations (Na+, K+, Mg2+, Ca2+, Mn2+) are stretched on hydrophobic surfaces, and directly observed by fluorescence microscopy. The results indicate that when these cations are added into the DNA solution, the fluorescence intensities of the stained DNA are reduced differently. The monovalent cations (Na+, K+) inhibit binding of histone to DNA. The divalent cations (Mg2+, Ca2+, Mn2+) enhance significantly the binding of histone to DNA and the binding of the DNA-histone complex to the hydrophobic surface. Mn2+ also induces condensation and aggregation of the DNA- histone complex.

  9. Determining the Architecture of a Protein-DNA Complex by Combining FeBABE Cleavage Analyses, 3-D Printed Structures, and the ICM Molsoft Program.

    Science.gov (United States)

    James, Tamara; Hsieh, Meng-Lun; Knipling, Leslie; Hinton, Deborah

    2015-01-01

    Determining the structure of a protein-DNA complex can be difficult, particularly if the protein does not bind tightly to the DNA, if there are no homologous proteins from which the DNA binding can be inferred, and/or if only portions of the protein can be crystallized. If the protein comprises just a part of a large multi-subunit complex, other complications can arise such as the complex being too large for NMR studies, or it is not possible to obtain the amounts of protein and nucleic acids needed for crystallographic analyses. Here, we describe a technique we used to map the position of an activator protein relative to the DNA within a large transcription complex. We determined the position of the activator on the DNA from data generated using activator proteins that had been conjugated at specific residues with the chemical cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). These analyses were combined with 3-D models of the available structures of portions of the activator protein and B-form DNA to obtain a 3-D picture of the protein relative to the DNA. Finally, the Molsoft program was used to refine the position, revealing the architecture of the protein-DNA within the transcription complex. PMID:26404142

  10. Ultra-low Doping on Two-Dimensional Transition Metal Dichalcogenides using DNA Nanostructure Doped by a Combination of Lanthanide and Metal Ions

    Science.gov (United States)

    Kang, Dong-Ho; Dugasani, Sreekantha Reddy; Park, Hyung-Youl; Shim, Jaewoo; Gnapareddy, Bramaramba; Jeon, Jaeho; Lee, Sungjoo; Roh, Yonghan; Park, Sung Ha; Park, Jin-Hong

    2016-02-01

    Here, we propose a novel DNA-based doping method on MoS2 and WSe2 films, which enables ultra-low n- and p-doping control and allows for proper adjustments in device performance. This is achieved by selecting and/or combining different types of divalent metal and trivalent lanthanide (Ln) ions on DNA nanostructures, using the newly proposed concept of Co-DNA (DNA functionalized by both divalent metal and trivalent Ln ions). The available n-doping range on the MoS2 by Ln-DNA is between 6 × 109 and 2.6 × 1010 cm-2. The p-doping change on WSe2 by Ln-DNA is adjusted between -1.0 × 1010 and -2.4 × 1010 cm-2. In Eu3+ or Gd3+-Co-DNA doping, a light p-doping is observed on MoS2 and WSe2 (~1010 cm-2). However, in the devices doped by Tb3+ or Er3+-Co-DNA, a light n-doping (~1010 cm-2) occurs. A significant increase in on-current is also observed on the MoS2 and WSe2 devices, which are, respectively, doped by Tb3+- and Gd3+-Co-DNA, due to the reduction of effective barrier heights by the doping. In terms of optoelectronic device performance, the Tb3+ or Er3+-Co-DNA (n-doping) and the Eu3+ or Gd3+-Co-DNA (p-doping) improve the MoS2 and WSe2 photodetectors, respectively. We also show an excellent absorbing property by Tb3+ ions on the TMD photodetectors.

  11. Study the effects of divalent metallic ions on the combination of DNA and histones with fluorescence anisotropy assays

    Institute of Scientific and Technical Information of China (English)

    LIU YuYing; WANG PengYe; DOU ShuoXing; XIE Ping; XI XuGuang

    2007-01-01

    The effects of divalent ions (Mn2+, Mg2+, Ca2+) on the interaction between DNA and histone are studied using a fluorescence anisotropy assay. Fluorescence anisotropies of DNA and DNA-histone in the presence of divalent ions (Mn2+, Mg2+, Ca2+) are measured. The results indicate that histone reduces the fluorescence anisotropy of lambda DNA while the divalent ions (Mn2+, Mg2+, Ca2+) significantly enhance the fluorescence anisotropy. Compared to the case of DNA incubated with histone alone, there are more histones binding to DNA when divalent ion, histone and DNA are incubated together. We also find that Mn2+ makes the DNA-histone complexes more condensed than the other ions do.

  12. Construction of a laser combiner for dual fluorescent single molecule imaging of pRNA of phi29 DNA packaging motor

    OpenAIRE

    Zhang, Hui; Shu, Dan; Browne, Mark,; Guo, Peixuan

    2009-01-01

    A customized laser combiner was designed and constructed for dual channel single molecule imaging. The feasibility of a combiner-incorporated imaging system was demonstrated in studies of single molecule FRET. Distance rulers made of dual-labeled dsDNA were used to evaluate the system by determining the distance between one FRET pair. The results showed that the system is sensitive enough to distinguish between distances differing by two base pair and the distances calculated from FRET effici...

  13. Use of vibrational spectroscopy to study protein and DNA structure, hydration, and binding of biomolecules: A combined theoretical and experimental approach

    DEFF Research Database (Denmark)

    Jalkanen, Karl J.; Jürgensen, Vibeke Würtz; Claussen, Anetta;

    2006-01-01

    We report on our work with vibrational absorption, vibrational circular dichroism, Raman scattering, Raman optical activity, and surface-enhanced Raman spectroscopy to study protein and DNA structure, hydration, and the binding of ligands, drugs, pesticides, or herbicides via a combined theoretic...

  14. Individual and combined effects of ochratoxin A and citrinin on viability and DNA fragmentation in cultured Vero cells and on chromosome aberrations in mice bone marrow cells

    International Nuclear Information System (INIS)

    Ochratoxin A (OTA) and citrinin (CTN) are two common contaminant mycotoxins which can occur jointly in a wide range of food commodities. Both mycotoxins have several toxic effects but share a significant nephrotoxic and carcinogenic potential since OTA and CTN were reported to be responsible for naturally occurring human and animal kidney diseases and tumors. Considering the concomitant production of OTA and CTN, it is very likely that humans and animals are always exposed to the mixture rather than to individual compounds. Therefore, the aim of the present study was to investigate, in vivo and in vitro, whether DNA damage is enhanced by combination of both mycotoxins as compared to their effect separately. To this end, we have assessed their effects individually or combined on cell proliferation and DNA fragmentation in cultured Vero cells and in vivo by monitoring the induction of chromosome aberrations. Our results clearly showed that cultured renal cells respond to OTA and CTN exposure by a moderate and weak inhibition of cell proliferation, respectively. However, when combined, they exert a significant increase in inhibition of cell viability. Similar results were found for the investigated genotoxicity endpoints (DNA fragmentation and chromosome aberrations). Altogether, our study showed that OTA and CTN combination effects are clearly synergistic. The synergistic induction of DNA damage observed with OTA and CTN taken concomitantly could be relevant to explain the molecular basis of the renal diseases and tumorogenesis induced by naturally occurring mycotoxins

  15. A combined DNA vaccine encoding BCSP31, SOD, and L7/L12 confers high protection against Brucella abortus 2308 by inducing specific CTL responses.

    Science.gov (United States)

    Yu, Da-Hai; Hu, Xi-Dan; Cai, Hong

    2007-06-01

    We constructed a combined DNA vaccine comprising genes encoding the antigens BCSP31, superoxide dismutase (SOD), and L7/L12 and evaluated its immunogenicity and protective efficacy. Immunization of mice with the combined DNA vaccine offered high protection against Brucella abortus (B. abortus) infection. The vaccine induced a vigorous specific immunoglobulin G (IgG) response, with higher IgG2a than IgG1 titers. Cytokine profiling performed at the same time showed a biased Th1-type immune response with significantly increased interferon-gamma and tumor necrosis factor-alpha stimulation. CD8(+), but not CD4(+), T cells accumulated at significantly higher levels after administration of the vaccine. Granzyme B-producing CD8(+) T cells were significantly higher in number in samples prepared from combined DNA-vaccinated mice compared with S19-vaccinated mice, demonstrating that the cytotoxicity lysis pathway is involved in the response to Brucella infection. The success of our combined DNA vaccine in a mouse model suggests its potential efficacy against brucellosis infection in large animals. PMID:17570767

  16. A combined DFT/Green’s function study on electrical conductivity through DNA duplex between Au electrodes

    OpenAIRE

    Tsukamoto, Takayuki; Ishikawa, Yasuyuki; Sengoku, Yasuo; Kurita, Noriyuki

    2009-01-01

    Electrical conducting properties of DNA duplexes sandwiched between Au electrodes have been investigated by use of first-principles molecular simulation based on DFT and Green’s function to elucidate the origin of their base sequence dependence. The theoretically simulated effects of DNA base sequence on the electrical conducting properties are in qualitative agreement with experiment. The HOMOs localized on Guanine bases have the major contribution to the electrical conductivity through DNA ...

  17. A combined DFT/Green’s function study on electrical conductivity through DNA duplex between Au electrodes

    Science.gov (United States)

    Tsukamoto, Takayuki; Ishikawa, Yasuyuki; Sengoku, Yasuo; Kurita, Noriyuki

    2009-06-01

    Electrical conducting properties of DNA duplexes sandwiched between Au electrodes have been investigated by use of first-principles molecular simulation based on DFT and Green's function to elucidate the origin of their base sequence dependence. The theoretically simulated effects of DNA base sequence on the electrical conducting properties are in qualitative agreement with experiment. The HOMOs localized on Guanine bases have the major contribution to the electrical conductivity through DNA duplexes.

  18. Mechanisms of immunotherapeutic intervention by anti-CD40L (CD154) antibody in an animal model of multiple sclerosis

    NARCIS (Netherlands)

    Howard, L.M.; Miga, A.J.; Vanderlugt, C.L.; Dal Canto, M.C.; Laman, J.D.; Noelle, R.J.; Miller, S.D.

    1999-01-01

    Relapsing experimental autoimmune encephalomyelitis (R-EAE) in the SJL mouse is a Th1-mediated autoimmune demyelinating disease model for human multiple sclerosis and is characterized by infiltration of the central nervous system (CNS) by Th1 cells and macrophages. Disease relapses are mediated by T

  19. Pay more attention to the immunologically comparative evaluation of HIV-1 DNA vaccine in combination with adjuvant cytokines: a long way to go

    Institute of Scientific and Technical Information of China (English)

    REN Xiao-feng

    2006-01-01

    @@ To the Editor: A recent article entitled "HIV-1 DNA vaccine with adjuvant cytokines induces specific immune responses against HIV-1 infection in mice"was published in the Chinese Medical Journal.1 The authors descriptively analyzed the adjuvant potential of an interleukin-12 (IL-12), interleukin-18 (IL-18),regulated on activation of normal T-cell expressed and secreted factor (RANTES), macrophage inflammatory protein 1 alpha (MIP-1α) or stromal cell derived factor 1 (SDF-1) plasmid when coimmunized with an HIV Gag gene-based DNA vaccine. Consequently the authors believed that IL-12, IL-18, or SDF-1 plasmid DNA potentially serves as immune adjuvants in combination with therapeutic HIV-1 DNA vaccine. The RANTES,MIP-1α plasmid DNA potentially serves as immune adjuvants in combination with HIV-1 prophylactic vaccine. Although the experiment allows us to consider such a coinoculation strategy as one of the means against HIV-1 infection, some special attentions should be highlighted.

  20. Synergistic anti-breast cancer effect of a combined treatment with the methyl donor S-adenosyl methionine and the DNA methylation inhibitor 5-aza-2'-deoxycytidine.

    Science.gov (United States)

    Chik, Flora; Machnes, Ziv; Szyf, Moshe

    2014-01-01

    DNA-demethylating agents activate tumor suppressor genes that are silenced by DNA methylation in cancer and are therefore emerging as a novel approach to cancer therapy. 5-azacytidine (VIDAZA), the first representative of this class of drugs was approved for treatment of myelodysplastic syndromes and is currently being tested on other cancers including solid tumors. However, 5-azacytidine or its deoxy-analog, 5-aza-2'-deoxycytidine (5-azaCdR) could also induce methylated prometastatic genes by DNA demethylation and induce cancer cell invasiveness. Since 5-azacytidine is a potent cancer growth inhibitor, we tested whether combining it with a DNA-methylating agent, the methyl donor S-adenosyl methionine (SAM), would block the adverse demethylating activity of 5-azaCdR while maintaining its growth suppression effects. We show here using several invasive and non-invasive breast cancer cell lines that SAM inhibits global- and gene-specific demethylation induced by 5-azaCdR, prevents 5-azaCdR activation of prometastatic genes uPA and MMP2, resulting in inhibition of cell invasiveness while augmenting the growth inhibitory effects of 5-azaCdR and its effects on tumor suppressor genes. Combination of drugs acting on the DNA methylation machinery at different levels is proposed as a new strategy for epigenetic therapy of cancer.

  1. Combined stimulation of IL-2 and 4-1BB receptors augments the antitumor activity of E7 DNA vaccines by increasing Ag-specific CTL responses.

    Science.gov (United States)

    Kim, Ha; Kwon, Byungsuk; Sin, Jeong-Im

    2013-01-01

    Human papillomavirus (HPV) infection is a major cause of cervical cancer. Here, we investigate whether concurrent therapy using HPV E7 DNA vaccines (pE7) plus IL-2 vs. IL-15 cDNA and anti-4-1BB Abs might augment antitumor activity against established tumors. IL-2 cDNA was slightly better than IL-15 cDNA as a pE7 adjuvant. Co-delivery of pE7+IL-2 cDNA increased tumor cure rates from 7% to 27%, whereas co-delivery of pE7+IL-2 cDNA with anti-4-1BB Abs increased tumor cure rates from 27% to 67% and elicited long-term memory responses. This increased activity was concomitant with increased induction of Ag-specific CTL activity and IFN-γ responses, but not with Ag-specific IgG production. Moreover, the combined stimulation of IL-2 and 4-1BB receptors with rIL-2 and anti-4-1BB Abs resulted in enhanced production of IFN-γ from Ag-specific CD8+ T cells. However, this effect was abolished by treatment with anti-IL-2 Abs and 4-1BB-Fc, suggesting that the observed effect was IL-2- and anti-4-1BB Ab-specific. A similar result was also obtained for Ag-specific CTL activity. Thus, these studies demonstrate that combined stimulation through the IL-2 and 4-1BB receptors augments the Ag-specific CD8+ CTL responses induced by pE7, increasing tumor cure rates and long-term antitumor immune memory. These findings may have implications for the design of DNA-based therapeutic vaccines against cancer. PMID:24391824

  2. Combined stimulation of IL-2 and 4-1BB receptors augments the antitumor activity of E7 DNA vaccines by increasing Ag-specific CTL responses.

    Directory of Open Access Journals (Sweden)

    Ha Kim

    Full Text Available Human papillomavirus (HPV infection is a major cause of cervical cancer. Here, we investigate whether concurrent therapy using HPV E7 DNA vaccines (pE7 plus IL-2 vs. IL-15 cDNA and anti-4-1BB Abs might augment antitumor activity against established tumors. IL-2 cDNA was slightly better than IL-15 cDNA as a pE7 adjuvant. Co-delivery of pE7+IL-2 cDNA increased tumor cure rates from 7% to 27%, whereas co-delivery of pE7+IL-2 cDNA with anti-4-1BB Abs increased tumor cure rates from 27% to 67% and elicited long-term memory responses. This increased activity was concomitant with increased induction of Ag-specific CTL activity and IFN-γ responses, but not with Ag-specific IgG production. Moreover, the combined stimulation of IL-2 and 4-1BB receptors with rIL-2 and anti-4-1BB Abs resulted in enhanced production of IFN-γ from Ag-specific CD8+ T cells. However, this effect was abolished by treatment with anti-IL-2 Abs and 4-1BB-Fc, suggesting that the observed effect was IL-2- and anti-4-1BB Ab-specific. A similar result was also obtained for Ag-specific CTL activity. Thus, these studies demonstrate that combined stimulation through the IL-2 and 4-1BB receptors augments the Ag-specific CD8+ CTL responses induced by pE7, increasing tumor cure rates and long-term antitumor immune memory. These findings may have implications for the design of DNA-based therapeutic vaccines against cancer.

  3. Combined DNA and lipid analyses of sediments reveal changes in Holocene phytoplankton populations in an Antarctic lake

    NARCIS (Netherlands)

    Sinninghe Damsté, J.S.; Coolen, M.J.L.; Muyzer, G.; Rijpstra, W.I.C.; Schouten, S.; Volkman, J.K.

    2004-01-01

    Preserved ribosomal DNA of planktonic phototrophic algae was recovered from Holocene anoxic sediments of Ace Lake (Antarctica), and the ancient community members were identified based on comparative sequence analysis. The similar concentration profiles of DNA of haptophytes and their traditional lip

  4. Oxidized dNTPs and the OGG1 and MUTYH DNA glycosylases combine to induce CAG/CTG repeat instability.

    Science.gov (United States)

    Cilli, Piera; Ventura, Ilenia; Minoprio, Anna; Meccia, Ettore; Martire, Alberto; Wilson, Samuel H; Bignami, Margherita; Mazzei, Filomena

    2016-06-20

    DNA trinucleotide repeat (TNR) expansion underlies several neurodegenerative disorders including Huntington's disease (HD). Accumulation of oxidized DNA bases and their inefficient processing by base excision repair (BER) are among the factors suggested to contribute to TNR expansion. In this study, we have examined whether oxidation of the purine dNTPs in the dNTP pool provides a source of DNA damage that promotes TNR expansion. We demonstrate that during BER of 8-oxoguanine (8-oxodG) in TNR sequences, DNA polymerase β (POL β) can incorporate 8-oxodGMP with the formation of 8-oxodG:C and 8-oxodG:A mispairs. Their processing by the OGG1 and MUTYH DNA glycosylases generates closely spaced incisions on opposite DNA strands that are permissive for TNR expansion. Evidence in HD model R6/2 mice indicates that these DNA glycosylases are present in brain areas affected by neurodegeneration. Consistent with prevailing oxidative stress, the same brain areas contained increased DNA 8-oxodG levels and expression of the p53-inducible ribonucleotide reductase. Our in vitro and in vivo data support a model where an oxidized dNTPs pool together with aberrant BER processing contribute to TNR expansion in non-replicating cells. PMID:26980281

  5. Rapid, highly sensitive and highly specific gene detection by combining enzymatic amplification and DNA chip detection simultaneously

    Directory of Open Access Journals (Sweden)

    Koji Hashimoto

    2016-05-01

    Full Text Available We have developed a novel gene detection method based on the loop-mediated isothermal amplification (LAMP reaction and the DNA dissociation reaction on the same DNA chip surface to achieve a lower detection limit, broader dynamic range and faster detection time than are attainable with a conventional DNA chip. Both FAM- and thiol-labeled DNA probe bound to the complementary sequence accompanying Dabcyl was immobilized on the gold surface via Au/thiol bond. The LAMP reaction was carried out on the DNA probe fixed gold surface. At first, Dabcyl molecules quenched the FAM fluorescence. According to the LAMP reaction, the complementary sequence with Dabcyl was competitively reacted with the amplified targeted sequence. As a result, the FAM fluorescence increased owing to dissociation of the complementary sequence from the DNA probe. The simultaneous reaction of LAMP and DNA chip detection was achieved, and 103 copies of the targeted gene were detected within an hour by measuring fluorescence intensity of the DNA probe.

  6. Combining combing and secondary ion mass spectrometry to study DNA on chips using 13C and 15N labeling

    Science.gov (United States)

    Cabin-Flaman, Armelle; Monnier, Anne-Francoise; Coffinier, Yannick; Audinot, Jean-Nicolas; Gibouin, David; Wirtz, Tom; Boukherroub, Rabah; Migeon, Henri-Noël; Bensimon, Aaron; Jannière, Laurent; Ripoll, Camille; Norris, Victor

    2016-01-01

    Dynamic secondary ion mass spectrometry ( D-SIMS) imaging of combed DNA – the combing, imaging by SIMS or CIS method – has been developed previously using a standard NanoSIMS 50 to reveal, on the 50 nm scale, individual DNA fibers labeled with different, non-radioactive isotopes in vivo and to quantify these isotopes. This makes CIS especially suitable for determining the times, places and rates of DNA synthesis as well as the detection of the fine-scale re-arrangements of DNA and of molecules associated with combed DNA fibers. Here, we show how CIS may be extended to 13C-labeling via the detection and quantification of the 13C 14N - recombinant ion and the use of the 13C: 12C ratio, we discuss how CIS might permit three successive labels, and we suggest ideas that might be explored using CIS. PMID:27429742

  7. Transient B-cell depletion with anti-CD20 in combination with proinsulin DNA vaccine or oral insulin: immunologic effects and efficacy in NOD mice.

    Directory of Open Access Journals (Sweden)

    Ghanashyam Sarikonda

    Full Text Available A recent type 1 diabetes (T1D clinical trial of rituximab (a B cell-depleting anti-CD20 antibody achieved some therapeutic benefit in preserving C-peptide for a period of approximately nine months in patients with recently diagnosed diabetes. Our previous data in the NOD mouse demonstrated that co-administration of antigen (insulin with anti-CD3 antibody (a T cell-directed immunomodulator offers better protection than either entity alone, indicating that novel combination therapies that include a T1D-related autoantigen are possible. To accelerate the identification and development of novel combination therapies that can be advanced into the clinic, we have evaluated the combination of a mouse anti-CD20 antibody with either oral insulin or a proinsulin-expressing DNA vaccine. Anti-CD20 alone, given once or on 4 consecutive days, produced transient B cell depletion but did not prevent or reverse T1D in the NOD mouse. Oral insulin alone (twice weekly for 6 weeks was also ineffective, while proinsulin DNA (weekly for up to 12 weeks showed a trend toward modest efficacy. Combination of anti-CD20 with oral insulin was ineffective in reversing diabetes in NOD mice whose glycemia was controlled with SC insulin pellets; these experiments were performed in three independent labs. Combination of anti-CD20 with proinsulin DNA was also ineffective in diabetes reversal, but did show modest efficacy in diabetes prevention (p = 0.04. In the prevention studies, anti-CD20 plus proinsulin resulted in modest increases in Tregs in pancreatic lymph nodes and elevated levels of proinsulin-specific CD4+ T-cells that produced IL-4. Thus, combination therapy with anti-CD20 and either oral insulin or proinsulin does not protect hyperglycemic NOD mice, but the combination with proinsulin offers limited efficacy in T1D prevention, potentially by augmentation of proinsulin-specific IL-4 production.

  8. One simple DNA extraction device and its combination with modified visual loop-mediated isothermal amplification for rapid on-field detection of genetically modified organisms.

    Science.gov (United States)

    Zhang, Miao; Liu, Yinan; Chen, Lili; Quan, Sheng; Jiang, Shimeng; Zhang, Dabing; Yang, Litao

    2013-01-01

    Quickness, simplicity, and effectiveness are the three major criteria for establishing a good molecular diagnosis method in many fields. Herein we report a novel detection system for genetically modified organisms (GMOs), which can be utilized to perform both on-field quick screening and routine laboratory diagnosis. In this system, a newly designed inexpensive DNA extraction device was used in combination with a modified visual loop-mediated isothermal amplification (vLAMP) assay. The main parts of the DNA extraction device included a silica gel membrane filtration column and a modified syringe. The DNA extraction device could be easily operated without using other laboratory instruments, making it applicable to an on-field GMO test. High-quality genomic DNA (gDNA) suitable for polymerase chain reaction (PCR) and isothermal amplification could be quickly isolated from plant tissues using this device within 15 min. In the modified vLAMP assay, a microcrystalline wax encapsulated detection bead containing SYBR green fluorescent dye was introduced to avoid dye inhibition and cross-contaminations from post-LAMP operation. The system was successfully applied and validated in screening and identification of GM rice, soybean, and maize samples collected from both field testing and the Grain Inspection, Packers, and Stockyards Administration (GIPSA) proficiency test program, which demonstrated that it was well-adapted to both on-field testing and/or routine laboratory analysis of GMOs.

  9. Allelic frequency distributions of 21 non-combined DNA index system STR loci in a Russian ethnic minority group from Inner Mongolia, China*

    OpenAIRE

    Wang, Hong-Dan; Shen, Chun-Mei; Liu, Wen-Juan; Zhang, Yu-Dang; Yang, Guang; Yan, Jiang-wei; Qin, Hai-Xia; Zhu, Bo-Feng

    2013-01-01

    We studied the allelic frequency distributions and statistical forensic parameters of 21 new short tandem repeat (STR) loci and the amelogenin locus, which are not included in the combined DNA index system (CODIS), in a Russian ethnic minority group from the Inner Mongolia Autonomous Region, China. A total of 114 bloodstain samples from unrelated individuals were extracted and co-amplified with four fluorescence-labeled primers in a multiplex polymerase chain reaction (PCR) system. Using capi...

  10. THE CLONING OF HRNT-1 USING A COMBINATION OF cDNA LIBRARY SCREENING WITH BIOTIN-LABELED PROBE AND RAPID AMPLIFICATION OF cDNA ENDS

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Kai-tai

    2001-01-01

    [1]Tom S, Andrew PR. Human Molecular Genetics [M]. John Wiley & Sons, Inc. United States of America 1996; 335.[2]Zhao Yong-liang, Jin Cui-zhen, Wu De-chang et al. Neoplastic transformation and cytogenetic changes of rat tracheal epithelial cells induced by a-particles irradiation [J]. Chin Med Sci J 1997; 12:202.[3]Frohman MA. Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE [J]. Methods Enzymol 1993; 218:340.[4]Frederick A, Roger B. Current Protocols in Molecular Biology [M]. John Wiley & Sons, Inc. United States of America 1998; 2.1.1.[5]Roux KH. Optimization and troubleshooting in PCR [J]. PCR Methods Appl 1995; 4:5158.[6]Sambrook, J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual [M]. 2nd Ed. New York: Cold Spring Harbor Laboratory, Cold Spring Harbor, 1989; 54.[7]Zhang Y, Frohman MA. Using rapid amplification of cDNA ends (RACE) to obtain full-length cDNAs [J]. Methods Mol Biol 1997; 69:61.[8]Frohman MA. Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE [J]. Methods Enzymol 1993; 218:340.[9]Iqbal S, Robinson J, Deere D, et al. Efficiency of the polymerase chain reaction amplification of the uid gene for detection of Escherichia coli in contaminated water [J]. Lett Appl Microbiol 1997; 24:498.[10]Schunck B, Kraft W, Truyen U. A simple touch-down polymerase chain reaction for the detection of canine parvovirus and feline panleukopenia virus in feces [J]. J Virol Methods 1995; 55:427.

  11. 日本血吸虫DNA疫苗联合免疫的研究进展%Research Progress on combined immunization of DNA vaccine against Schistosoma japonicum

    Institute of Scientific and Technical Information of China (English)

    冯其梅; 张树菊; 汪世平

    2012-01-01

    疫苗作为预防和控制传染病的重要手段,已成功的控制了很多传染病的流行.因此,许多研究者希望研制一种安全有效的血吸虫病疫苗,以期达到控制血吸虫病流行的目的.近年来,鉴于单价疫苗有限的免疫效果,人们开始进行日本血吸虫DNA疫苗联合免疫的研究.本文对日本血吸虫DNA疫苗联合免疫的研究资料进行了归纳整理,发现日本血吸虫DNA疫苗联合免疫涉及鸡尾酒式DNA混合疫苗、双价和多价DNA联合疫苗以及佐剂研究等方面,比较减虫卒、减卵率等指标,联合免疫疫苗的效果明显优于单价DNA疫苗的效果,而且具有潜在的实际应用价值.%As an important mean of prevention and control of infectious diseases, vaccine has been successfully took control. Therefore, in order to control the prevalence of sehistosomiasis japonica, many scientists want to develop a safe and effective vaccine against Schistosoma japonicum. Recently, in view of the limited immune effect of monovalent vaccine, people have started to study combined immunization of DNA vaccine of Schistosoma japonicum. This paper review the development of combined immunization of DNA vaccine against Schistosoma japonicum , and found that combined DNA vaccines include cocktail hybrid DNA vaccines, bivalent and multivalent DNA vaccines, and DNA vaccines with adjuvant mainly. They induced higher worm reduction rate and liver egg reduction rate, and would be well worthy of to be study.

  12. Arsenic-Induced Antioxidant Depletion, Oxidative DNA Breakage, and Tissue Damages are Prevented by the Combined Action of Folate and Vitamin B12.

    Science.gov (United States)

    Acharyya, Nirmallya; Deb, Bimal; Chattopadhyay, Sandip; Maiti, Smarajit

    2015-11-01

    Arsenic is a grade I human carcinogen. It acts by disrupting one-carbon (1C) metabolism and cellular methyl (-CH3) pool. The -CH3 group helps in arsenic disposition and detoxification of the biological systems. Vitamin B12 and folate, the key promoters of 1C metabolism were tested recently (daily 0.07 and 4.0 μg, respectively/100 g b.w. of rat for 28 days) to evaluate their combined efficacy in the protection from mutagenic DNA-breakage and tissue damages. The selected tissues like intestine (first-pass site), liver (major xenobiotic metabolizer) and lung (major arsenic accumulator) were collected from arsenic-ingested (0.6 ppm/same schedule) female rats. The hemo-toxicity and liver and kidney functions were monitored. Our earlier studies on arsenic-exposed humans can correlate carcinogenesis with DNA damage. Here, we demonstrate that the supplementation of physiological/therapeutic dose of vitamin B12 and folate protected the rodents significantly from arsenic-induced DNA damage (DNA fragmentation and comet assay) and hepatic and renal tissue degeneration (histo-architecture, HE staining). The level of arsenic-induced free-radical products (TBARS and conjugated diene) was significantly declined by the restored actions of several antioxidants viz. urate, thiol, catalase, xanthine oxidase, lactoperoxidase, and superoxide dismutase in the tissues of vitamin-supplemented group. The alkaline phosphatase, transaminases, urea and creatinine (hepatic and kidney toxicity marker), and lactate dehydrogenase (tissue degeneration marker) were significantly impaired in the arsenic-fed group. But a significant protection was evident in the vitamin-supplemented group. In conclusion, the combined action of folate and B12 results in the restitution in the 1C metabolic pathway and cellular methyl pool. The cumulative outcome from the enhanced arsenic methylation and antioxidative capacity was protective against arsenic induced mutagenic DNA breakages and tissue damages.

  13. Combined IL-12 Plasmid and Recombinant SjGST Enhance the Protective and Anti-pathology Effect of SjGST DNA Vaccine Against Schistosoma japonicum.

    Science.gov (United States)

    Cheng, Po-Ching; Lin, Ching-Nan; Peng, Shih-Yi; Kang, Tsung-Fu; Lee, Kin-Mu

    2016-02-01

    Schistosomiasis is listed as one of most important tropical diseases and more than 200 million people are estimated to be infected. Development of a vaccine is thought to be the most effective way to control this disease. Recombinant 26-kDa glutathione S-transferase (rSjGST) has previously been reported to achieve a worm reduction rate of 42-44%. To improve the efficiency of the vaccine against Schistosoma japonicum, we immunized mice with a combination of pcDNA vector-encoded 26-kDa SjGST (pcDNA/SjGST), IL-12 expressing-plasmid (pIL-12), and rSjGST. Co-vaccination with pcDNA/SjGST, pIL-12, and rSjGST led to a reduction in worm burden, hepatic egg burden, and the size of liver tissue granulomas than that in the untreated infection controls. In addition, we detected high levels of specific IgG, IgG1, and IgG2a against the rSjGST antigen in infected mice vaccinated with this combination of pcDNA/SjGST, pIL-12, and rSjGST. Moreover, high expression levels of Th2 cytokines, including IL-4 and IL-10, were also detected in this group, without diminished levels of IL-12, INF-γ, and TNF-α cytokines that are related to parasite killing. In conclusion, we have developed a new vaccination regimen against S. japonicum infection and shown that co-immunization with pcDNA/SjGST vaccine, pIL-12, and rSjGST has significant anti-parasite, anti-hepatic egg and anti-pathology effects in mice. The efficacy of this vaccination method should be further validated in large animals such as water buffalo. This method may help to reduce the transmission of zoonotic schistosomiasis japonica. PMID:26891172

  14. Targeting DNA repair with combination veliparib (ABT-888) and temozolomide in patients with metastatic castration-resistant prostate cancer

    OpenAIRE

    Hussain, Maha; Carducci, Michael A.; Slovin, Susan; Cetnar, Jeremy; Qian, Jiang; McKeegan, Evelyn M.; Refici-Buhr, Marion; Chyla, Brenda; Shepherd, Stacie P.; Giranda, Vincent L.; Alumkal, Joshi J.

    2014-01-01

    Androgen receptor-mediated transcription is directly coupled with the induction of DNA damage, and castration-resistant tumor cells exhibit increased activity of poly (ADP-ribose) polymerase (PARP)-1, a DNA repair enzyme. This study assessed the efficacy and safety of low dose oral PARP inhibitor veliparib (ABT-888) and temozolomide (TMZ) in docetaxel-pretreated patients with metastatic castration-resistant prostate cancer (mCRPC) in a single-arm, open-label, pilot study. Patients with mCRPC ...

  15. Combined treatment with vitamin B12b and ascorbic acid causes in vitro DNA degradation in tumor cells.

    Science.gov (United States)

    Medvedev, A I; Akatov, V S; Kreshchenko, N D; Solov'eva, M E; Leshchenko, V V; Lezhnev, E I; Yakubovskaya, R I

    2001-04-01

    Incubation of Ehrlich ascites carcinoma and HEp-2 human epidermoid laryngeal carcinoma cells with hydroxycobalamin (vitamin B12b) and ascorbic acid induced generation and accumulation of double-stranded DNA fragments (23,000 b.p. and longer) in cells. The same vitamins alone in the same concentrations produced no such effects. DNA degradation in HEp-2 cells caused by long-term (4 h) incubation with 5-25 microM hydroxycobalamin and ascorbic acid (1:10-1:40 molar ratio) at 37 degrees C was comparable with that induced by gamma-irradiation in a dose of 150 Gy at 4 degrees C.

  16. The combined effects of BDE47 and BaP on oxidatively generated DNA damage in L02 cells and the possible molecular mechanism.

    Science.gov (United States)

    An, Jing; Yin, Lingling; Shang, Yu; Zhong, Yufang; Zhang, Xinyu; Wu, Minghong; Yu, Zhiqiang; Sheng, Guoying; Fu, Jiamo; Huang, Yuecheng

    2011-04-01

    Polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs) coexist widely in the environment and have generated adverse effects on the environment and human health. The purpose of this study was to investigate the combined toxic effects of these chemicals and the related mechanism. L02 cells were exposed to BDE47 (5, 10μmol/L) or/and BaP (50μmol/L) in different administration order. The cell growth and survival, DNA strand breaks, oxidative stress index (ROS, SOD, GSH, and MDA), LDH release and the expression level of CYP1 family members were measured. The result showed that BDE47 or/and BaP had no effect on the cell growth and survival under the present conditions. However, compared with the groups treated with BDE47 or BaP alone, the combined-treated groups induced significantly elevated DNA strand breaks, ROS production, and MDA level. Especially, pretreatment with BDE47 followed by BaP led to the strongest effects. Addition of the antioxidant N-acetyl-l-cysteine (NAC) markedly reduced the ROS level and partly suppressed the DNA strand breaks induced by BDE47 or/and BaP. Meanwhile, the combined treatment groups also markedly increased the SOD activity, GSH content, and LDH release level compared with the control group. The real-time PCR results showed that BaP could significantly induce the expression of CYP1A1 and CYP1B1, however, the pre-treatment with BDE47 appeared to attenuate the BaP-induced CYP1 expression. All of above findings indicated that BDE47 and BaP had a synergistic effect on oxidatively generated DNA damage in L02 cells via regulation on the oxidative stress response and the expression of CYP1 metabolism enzymes.

  17. Features of the Arabidopsis recombination landscape resulting from the combined loss of sequence variation and DNA methylation

    NARCIS (Netherlands)

    Colomé Tatché, Maria; Cortijo, Sandra; Wardenaar, Rene; Monteiro Morgado, Lionel; Lahouze, Benoit; Sarazin, Alexis; Etcheverry, Mathilde; Martin, Antoine; Feng, Suhua; Duvernois-Berthet, Evelyne; Labadie, Karine; Wincker, Patrick; Jacobsen, Steven E.; Jansen, Ritsert C.; Colot, Vincent; Johannes, Frank

    2012-01-01

    The rate of meiotic crossing over (CO) varies considerably along chromosomes, leading to marked distortions between physical and genetic distances. The causes underlying this variation are being unraveled, and DNA sequence and chromatin states have emerged as key factors. However, the extent to whic

  18. Membrane Destruction and DNA Binding of Staphylococcus aureus Cells Induced by Carvacrol and Its Combined Effect with a Pulsed Electric Field.

    Science.gov (United States)

    Wang, Lang-Hong; Wang, Man-Sheng; Zeng, Xin-An; Zhang, Zhi-Hong; Gong, De-Ming; Huang, Yan-Bo

    2016-08-17

    Carvacrol (5-isopropyl-2-methylphenol, CAR) is an antibacterial ingredient that occurs naturally in the leaves of the plant Origanum vulgare. The antimicrobial mechanism of CAR against Staphylococcus aureus ATCC 43300 was investigated in the study. Analysis of the membrane fatty acids by gas chromatography-mass spectrometry (GC-MS) showed that exposure to CAR at low concentrations induced a marked increase in the level of unbranched fatty acids (from 34.90 ± 1.77% to 62.37 ± 4.26%). Moreover, CAR at higher levels severely damaged the integrity and morphologies of the S. aureus cell membrane. The DNA-binding properties of CAR were also investigated using fluorescence, circular dichroism, molecular modeling, and atomic-force microscopy. The results showed that CAR bound to DNA via the minor-groove mode, mildly perturbed the DNA secondary structure, and induced DNA molecules to be aggregated. Furthermore, a combination of CAR with a pulsed-electric field was found to exhibit strong synergistic effects on S. aureus. PMID:27420472

  19. Development of a 24-locus multiplex system to incorporate the core loci in the Combined DNA Index System (CODIS) and the European Standard Set (ESS).

    Science.gov (United States)

    Guo, Fei; Shen, Hongying; Tian, Huaizhou; Jin, Ping; Jiang, Xianhua

    2014-01-01

    The 24-locus multiplex system allows co-amplification and fluorescent detection of 24 loci (23 STR loci and Amelogenin), including STR loci in the Combined DNA Index System (CODIS) and the ESS (European Standard Set) as well as five additional loci (D2S1338, D6S1043, D19S433, Penta D and Penta E) commonly used in commercial kits. It facilitates data sharing and minimizes adventitious matches within national or between international DNA databases. Additionally, the system can amplify directly from blood and buccal samples spotted on filter paper and swabs and reduce the cycling time to less than one hour and a half. Primers, internal size standard, allelic ladders and matrix standard set were designed and created in-house with a design strategy to work in this multiplex. Developmental validation experiments followed the Scientific Working Group on DNA Analysis Methods (SWGDAM) and the Chinese National Standard (GA/T815-2009) guidelines. The system was evaluated by species specificity, sensitivity, stability, precision and accuracy, case-type samples, population, mixture and PCR-based studies. The results demonstrate that the 24-locus multiplex system is a robust and reliable identification assay as required for forensic DNA typing and databasing.

  20. Membrane Destruction and DNA Binding of Staphylococcus aureus Cells Induced by Carvacrol and Its Combined Effect with a Pulsed Electric Field.

    Science.gov (United States)

    Wang, Lang-Hong; Wang, Man-Sheng; Zeng, Xin-An; Zhang, Zhi-Hong; Gong, De-Ming; Huang, Yan-Bo

    2016-08-17

    Carvacrol (5-isopropyl-2-methylphenol, CAR) is an antibacterial ingredient that occurs naturally in the leaves of the plant Origanum vulgare. The antimicrobial mechanism of CAR against Staphylococcus aureus ATCC 43300 was investigated in the study. Analysis of the membrane fatty acids by gas chromatography-mass spectrometry (GC-MS) showed that exposure to CAR at low concentrations induced a marked increase in the level of unbranched fatty acids (from 34.90 ± 1.77% to 62.37 ± 4.26%). Moreover, CAR at higher levels severely damaged the integrity and morphologies of the S. aureus cell membrane. The DNA-binding properties of CAR were also investigated using fluorescence, circular dichroism, molecular modeling, and atomic-force microscopy. The results showed that CAR bound to DNA via the minor-groove mode, mildly perturbed the DNA secondary structure, and induced DNA molecules to be aggregated. Furthermore, a combination of CAR with a pulsed-electric field was found to exhibit strong synergistic effects on S. aureus.

  1. Visual recovery in a man with the rare combination of mtDNA 11778 LHON mutation and a MS-like disease after mitoxantrone therapy.

    Science.gov (United States)

    Buhmann, C; Gbadamosi, J; Heesen, C

    2002-10-01

    We describe a young man with prognostic unfavourable homoplasmatic mitochondrial DNA(mt DNA) 11778 Leber's hereditary optic neuropathy (LHON) point mutation and confirmed multiple sclerosis (MS). This combination of LHON and MS-like disease is rare in both sexes, and in men has been described in only a few case reports. In a 4-year follow-up during immunosuppressive therapy with mitoxantrone, we found a remarkable time delayed visual recovery 12 months after acute onset of rapid sequential bilateral subtotal visual loss followed by episodes of isolated acute demyelinative optic neuropathy. Visual recovery to such extent after this latency is uncommon in both mtDNA 11778 LHON mutation and optic neuritis (ON) in MS. Relapses in visual deterioration must be considered as extremely rare in LHON. This case might support the hypothesis of an immunological pathogenetic factor in combined LHON and MS, and possibly in LHON alone. We suggest a search for the LHON mutation in MS patients with predominant visual impairment, independent of patients' gender.

  2. Combination of 768-well microplate array diagonal gel electrophoresis with duplex PCR of X and Y chromosome markers for quality control of epidemiological DNA banks.

    Science.gov (United States)

    Huang, Shuwen; Chen, Xiao-he; Day, Ian N M

    2006-08-01

    Large DNA banks for human epidemiological studies have become an increasingly important research tool. The power of genotype-phenotype studies is dependent both on the quality of phenotyping and of genotyping and of correct linking of phenotypes to genotypes. Samples must be tracked through numerous steps between subject or patient and post-genotypic data. Only one phenotype, sex, has a perfect and binary correlation with genotype. In mixed sex studies, it may be advantageous for purposes of quality control to keep sexes mixed during the steps from acquisition to DNA bank, in order to be able to check later for sample swaps. We have designed a duplex PCR combining an amplicon from MAOA marking the X chromosome and an amplicon from DDX3Y marking the Y chromosome. We combined this with a simple economical palmtop sized 768-well microplate compatible electrophoresis system developed in-house for examination of duplex PCR products. We applied this quality control test in the validation of two DNA banks.

  3. Enhanced anti-tumor effect of zoledronic acid combined with temozolomide against human malignant glioma cell expressing O6-methylguanine DNA methyltransferase.

    Directory of Open Access Journals (Sweden)

    Junya Fukai

    Full Text Available Temozolomide (TMZ, a DNA methylating agent, is widely used in the adjuvant treatment of malignant gliomas. O6-methylguanine-DNA methyltranferase (MGMT, a DNA repair enzyme, is frequently discussed as the main factor that limits the efficacy of TMZ. Zoledronic acid (ZOL, which is clinically applied to treat cancer-induced bone diseases, appears to possess direct anti-tumor activity through apoptosis induction by inhibiting mevalonate pathway and prenylation of intracellular small G proteins. In this study, we evaluated whether ZOL can be effectively used as an adjuvant to TMZ in human malignant glioma cells that express MGMT. Malignant glioma cell lines, in which the expression of MGMT was detected, did not exhibit growth inhibition by TMZ even at a longer exposure. However, combination experiment of TMZ plus ZOL revealed that a supra-additive effect resulted in a significant decrease in cell growth. In combined TMZ/ZOL treatment, an increased apoptotic rate was apparent and significant activation of caspase-3 and cleavage of poly-(ADP-ribose polymerase were observed compared with each single drug exposure. There were decreased amounts of Ras-GTP, MAPK and Akt phosphorylation and MGMT expression in the ZOL-treated cells. Subcutanous xenograft models showed significant decrease of tumor growth with combined TMZ/ZOL treatment. These results suggest that ZOL efficaciously inhibits activity of Ras in malignant glioma cells and potentiates TMZ-mediated cytotoxicity, inducing growth inhibition and apoptosis of malignant glioma cells that express MGMT and resistant to TMZ. Based on this work, combination of TMZ with ZOL might be a potential therapy in malignant gliomas that receive less therapeutic effects of TMZ due to cell resistance.

  4. Predictive microbiology combined with metagenomic analysis targeted on the 16S rDNA : A new approach for food quality

    OpenAIRE

    Delhalle, Laurent; Ellouze, Mariem; Taminiau, Bernard; Korsak Koulagenko, Nicolas; Nezer, Carine

    2013-01-01

    OBJECTIVES The food spoilage process is mainly caused by alteration micro-organisms and classical culture-based methods have therefore been used to assess the microbiological quality of food. These techniques are simple to implement but may not be relevant to understand the modifications of the microbial ecology which occur in the food product in response to different changes in the environmental conditions. Metagenomic analysis targeted on 16S ribosomal DNA can bring about a solution to t...

  5. Evaluating the combinative effects on human lymphocyte DNA damage induced by Ultraviolet ray C plus 1.8 GHz microwaves using comet assay in vitro

    International Nuclear Information System (INIS)

    The objective of this study was to observe whether 1.8 GHz microwaves (MW) (SAR, 3 W/kg) exposure can influence human lymphocyte DNA damage induced by ultraviolet ray C (UVC). The lymphocytes, which were from three young healthy donors, were exposed to 254nm UVC at the doses of 0.25, 0.5, 0.75, 1.0, 1.5and 2.0 J m-2, respectively. The lymphocytes were irradiated by 1.8GHz MW (SAR, 3 W/kg) for 0, 1.5 and 4 h. The combinative exposure of UVC plus MW was conducted. The treated cells were incubated for 0, 1.5 and 4 h. Finally, comet assay was used to measure DNA damage of above treated lymphocytes. The results indicated that the difference of DNA damage induced between MW group and control group was not significant (P > 0.05). The MTLs induced by UVC were 1.71 ± 0.09, 2.02 ± 0.08, 2.27 ± 0.17, 2.27 ± 0.06, 2.25 ± 0.12, 2.24 ± 0.11 μm, respectively, which were significantly higher than that (0.96 ± 0.05 μm) of control (P < 0.01). MTLs of some sub-groups in combinative exposure groups at 1.5-h incubation were significantly lower that those of corresponding UVC sub-groups (P < 0.01 or P < 0.05). However, MTLs of some sub-groups in combinative exposure groups at 4-h incubation were significantly higher that those of corresponding UVC sub-groups (P < 0.01 or P < 0.05). In this experiment it was found that 1.8 GHz (SAR, 3 W/kg) MW exposure for 1.5 and 4 h did not enhance significantly human lymphocyte DNA damage, but could reduce and increase DNA damage of human lymphocytes induced by UVC at 1.5-h and 4-h incubation, respectively

  6. Combined IL-12 Plasmid and Recombinant SjGST Enhance the Protective and Anti-pathology Effect of SjGST DNA Vaccine Against Schistosoma japonicum.

    OpenAIRE

    Po-Ching Cheng; Ching-Nan Lin; Shih-Yi Peng; Tsung-Fu Kang; Kin-Mu Lee

    2016-01-01

    Schistosomiasis is listed as one of most important tropical diseases and more than 200 million people are estimated to be infected. Development of a vaccine is thought to be the most effective way to control this disease. Recombinant 26-kDa glutathione S-transferase (rSjGST) has previously been reported to achieve a worm reduction rate of 42–44%. To improve the efficiency of the vaccine against Schistosoma japonicum, we immunized mice with a combination of pcDNA vector-encoded 26-kDa SjGST (p...

  7. Inhibition of DNA Topoisomerase Type IIα (TOP2A by Mitoxantrone and Its Halogenated Derivatives: A Combined Density Functional and Molecular Docking Study

    Directory of Open Access Journals (Sweden)

    Md. Abu Saleh

    2016-01-01

    Full Text Available In this study, mitoxantrone and its halogenated derivatives have been designed by density functional theory (DFT to explore their structural and thermodynamical properties. The performance of these drugs was also evaluated to inhibit DNA topoisomerase type IIα (TOP2A by molecular docking calculation. Noncovalent interactions play significant role in improving the performance of halogenated drugs. The combined quantum and molecular mechanics calculations revealed that CF3 containing drug shows better preference in inhibiting the TOP2A compared to other modified drugs.

  8. Personal identification of cold case remains through combined contribution from anthropological, mtDNA and bomb–pulse dating analyses*†

    Science.gov (United States)

    Speller, Camilla F.; Spalding, Kirsty L.; Buchholz, Bruce A.; Hildebrand, Dean; Moore, Jason; Mathewes, Rolf; Skinner, Mark F.; Yang, Dongya Y.

    2013-01-01

    In 1968, a child’s cranium was recovered from the banks of a northern Canadian river, and held in trust until the ‘cold case’ was re-opened in 2005. The cranium underwent re-analysis at the Centre for Forensic Research, Simon Fraser University, using recently developed anthropological, ‘bomb-pulse’ radiocarbon analysis and forensic DNA techniques. Craniometrics, skeletal ossification and dental formation indicated an age-at-death of 4.4 ±1 years. Radiocarbon analysis of enamel from two teeth indicated a year of birth between 1958–1962. Forensic DNA analysis indicated the child was male, and the obtained mitochondrial profile matched a living maternal relative of the presumed missing child. These multi-disciplinary analyses resulted in a legal identification 41 years after the discovery of the remains, highlighting the enormous potential of combining radiocarbon analysis with anthropological and mtDNA analyses in producing confident personal identifications for forensic cold cases dating to within the last 60 years. PMID:22804335

  9. Response of arsenic-induced oxidative stress, DNA damage, and metal imbalance to combined administration of DMSA and monoisoamyl-DMSA during chronic arsenic poisoning in rats.

    Science.gov (United States)

    Bhadauria, S; Flora, S J S

    2007-03-01

    Arsenic and its compounds cause adverse health effects in humans. Current treatment employs administration of thiol chelators, such as meso-2,3-dimercaptosuccinic acid (DMSA) and sodium 2,3-dimercaptopropane 1-sulfonate (DMPS), which facilitate its excretion from the body. However, these chelating agents are compromised by number of limitations due to their lipophobic nature, particularly in case of chronic poisoning. Combination therapy is a new approach to ensure enhanced removal of metal from the body, reduced doses of potentially toxic chelators, and no redistribution of metal from one organ to another, following chronic metal exposure. The present study attempts to investigate dose-related effects of two thiol chelators, DMSA and one of its new analogues, monoisoamyl dimercaptosuccinic acid (MiADMSA), when administered in combination with the aim of achieving normalization of altered biochemical parameters suggestive of oxidative stress and depletion of inorganic arsenic following chronic arsenic exposure. Twenty-five adult male Wistar rats were given 25 ppm arsenic for 10 weeks followed by chelation therapy with the above chelating agents at a dose of 0.3 mmol/kg (orally) when administered individually or 0.15 mmol/kg and 0.3 mmol/kg (once daily for 5 consecutive days), respectively, when administered in combination. Arsenic exposure led to the inhibition of blood delta-aminolevulinic acid dehydratase (ALAD) activity and depletion of glutathione (GSH) level. These changes were accompanied by significant depletion of hemoglobin, RBC and Hct as well as blood superoxide dismutase (SOD) acitivity. There was an increase in hepatic and renal levels of thiobarbituric acid-reactive substances, while GSH:GSSG ratio decreased significantly, accompanied by a significant increase in metallothionein (MT) in hepatocytes. DNA damage based on denaturing polyacrylamide gel electrophoresis revealed significant loss in the integrity of DNA extracted from the liver of arsenic

  10. CD40 ligand and tdTomato-armed vaccinia virus for induction of antitumor immune response and tumor imaging.

    Science.gov (United States)

    Parviainen, S; Ahonen, M; Diaconu, I; Hirvinen, M; Karttunen, Å; Vähä-Koskela, M; Hemminki, A; Cerullo, V

    2014-02-01

    Oncolytic vaccinia virus is an attractive platform for immunotherapy. Oncolysis releases tumor antigens and provides co-stimulatory danger signals. However, arming the virus can improve efficacy further. CD40 ligand (CD40L, CD154) can induce apoptosis of tumor cells and it also triggers several immune mechanisms. One of these is a T-helper type 1 (Th1) response that leads to activation of cytotoxic T-cells and reduction of immune suppression. Therefore, we constructed an oncolytic vaccinia virus expressing hCD40L (vvdd-hCD40L-tdTomato), which in addition features a cDNA expressing the tdTomato fluorochrome for detection of virus, potentially important for biosafety evaluation. We show effective expression of functional CD40L both in vitro and in vivo. In a xenograft model of bladder carcinoma sensitive to CD40L treatment, we show that growth of tumors was significantly inhibited by the oncolysis and apoptosis following both intravenous and intratumoral administration. In a CD40-negative model, CD40L expression did not add potency to vaccinia oncolysis. Tumors treated with vvdd-mCD40L-tdtomato showed enhanced efficacy in a syngenic mouse model and induced recruitment of antigen-presenting cells and lymphocytes at the tumor site. In summary, oncolytic vaccinia virus coding for CD40L mediates multiple antitumor effects including oncolysis, apoptosis and induction of Th1 type T-cell responses.

  11. Correlation of in Situ Oxazolidine Formation with Highly Synergistic Cytotoxicity and DNA Cross-Linking in Cancer Cells from Combinations of Doxorubicin and Formaldehyde.

    Science.gov (United States)

    Barthel, Benjamin L; Mooz, Erin L; Wiener, Laura Elizabeth; Koch, Gary G; Koch, Tad H

    2016-03-10

    Anthracyclines are a class of antitumor compounds that are successful and widely used but suffer from cardiotoxicity and acquired tumor resistance. Formaldehyde interacts with anthracyclines to enhance antitumor efficacy, bypass resistance mechanisms, improve the therapeutic profile, and change the mechanism of action from a topoisomerase II poison to a DNA cross-linker. Contrary to current dogma, we show that both efficient DNA cross-linking and potent synergy in combination with formaldehyde correlate with the anthracycline's ability to form cyclic formaldehyde conjugates as oxazolidine moieties and that the cyclic conjugates are better cross-linking agents and cytotoxins than acyclic conjugates. We also provide evidence that suggests that the oxazolidine forms in situ, since cotreatment with doxorubicin and formaldehyde is highly cytotoxic to dox-resistant tumor cell lines, and that this benefit is absent in combinations of formaldehyde and epirubicin, which cannot form stable oxazolidines. These results have potential clinical implications in the active field of anthracycline prodrug design and development. PMID:26881291

  12. A Combined Computational and Experimental Study on the Structure-Regulation Relationships of Putative Mammalian DNA Replication Initiator GINS

    Institute of Scientific and Technical Information of China (English)

    Reiko Hayashi; Takako Arauchi; Moe Tategu; Yuya Goto; Kenichi Yoshida

    2006-01-01

    GINS, a heterotetramer of SLD5, PSF1, PSF2, and PSF3 proteins, is an emerging chromatin factor recognized to be involved in the initiation and elongation step of DNA replication. Although the yeast and Xenopus GINS genes are well documented, their orthologous genes in higher eukaryotes are not fully characterized.In this study, we report the genomic structure and transcriptional regulation of mammalian GINS genes. Serum stimulation increased the GINS Mrna levels in human cells. Reporter gene assay using putative GINS promoter sequences revealed that the expression of mammalian GINS is regulated by 17β-Estradiolstimulated estrogen receptor α, and human PSF3 acts as a gene responsive to transcription factor E2F1. The goal of this study is to present the current data so as to encourage further work in the field of GINS gene regulation and functions in mammalian cells.

  13. Blood levels of histone-complexed DNA fragments are associated with coagulopathy, inflammation and endothelial damage early after trauma

    Directory of Open Access Journals (Sweden)

    Pär I Johansson

    2013-01-01

    Full Text Available Background: Tissue injury increases blood levels of extracellular histones and nucleic acids, and these may influence hemostasis, promote inflammation and damage the endothelium. Trauma-induced coagulopathy (TIC may result from an endogenous response to the injury that involves the neurohumoral, inflammatory and hemostatic systems. Aims: To study the contribution of extracellular nucleic constituents to TIC, inflammation and endothelial damage. Setting and Design: Prospective observational study. Materials and Methods: We investigated histone-complexed DNA fragments (hcDNA along with biomarkers of coagulopathy, inflammation and endothelial damage in plasma from 80 trauma patients admitted directly to the Trauma Centre from the scene of the accident. Blood was sampled a median of 68 min (IQR 48-88 post injury. Trauma patients with hcDNA levels >median or ≤median were compared. Results: Trauma patients with high plasma hcDNA had higher Injury Severity Score (ISS and level of sympathoadrenal activation (higher adrenaline and noradrenaline and a higher proportion of prolonged activated partial thromboplastin time (APTT and higher D-dimer, tissue-type plasminogen activator (tPA, Annexin V and soluble CD40 ligand (sCD40L concurrent with lower plasminogen activator inhibitor (PAI-1 and prothrombin fragment (PF 1 + 2 (all P < 0.05, all indicative of impaired thrombin generation, hyperfibrinolysis and platelet activation. Furthermore, patients with high hcDNA had enhanced inflammation and endothelial damage evidenced by higher plasma levels of terminal complement complex (sC5b-9, IL-6, syndecan-1, thrombomodulin and tissue factor pathway inhibitor (all P < 0.05. Conclusions: Excessive release of extracellular histones and nucleic acids seems to contribute to the hypocoagulability, inflammation and endothelial damage observed early after trauma.

  14. A rapid and versatile combined DNA/RNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsis thaliana ecotypes Col-0 and Landsberg erecta

    Directory of Open Access Journals (Sweden)

    Coupland George

    2005-08-01

    Full Text Available Abstract Background Many established PCR-based approaches in plant molecular biology rely on lengthy and expensive methods for isolation of nucleic acids. Although several rapid DNA isolation protocols are available, they have not been tested for simultaneous RNA isolation for RT-PCR applications. In addition, traditional map-based cloning technologies often use ill-proportioned marker regions even when working with the model plant Arabidopsis thaliana, where the availability of the full genome sequence can now be exploited for the creation of a high-density marker systems. Results We designed a high-density polymorphic marker set between two frequently used ecotypes. This new polymorphic marker set allows size separation of PCR products on agarose gels and provides an initial resolution of 10 cM in linkage mapping experiments, facilitated by a rapid plant nucleic acid extraction protocol using minimal amounts of A. thaliana tissue. Using this extraction protocol, we have also characterized segregating T-DNA insertion mutations. In addition, we have shown that our rapid nucleic acid extraction protocol can also be used for monitoring transcript levels by RT-PCR amplification. Finally we have demonstrated that our nucleic acid isolation method is also suitable for other plant species, such as tobacco and barley. Conclusion To facilitate high-throughput linkage mapping and other genomic applications, our nucleic acid isolation protocol yields sufficient quality of DNA and RNA templates for PCR and RT-PCR reactions, respectively. This new technique requires considerably less time compared to other purification methods, and in combination with a new polymorphic PCR marker set dramatically reduces the workload required for linkage mapping of mutations in A. thaliana utilizing crosses between Col-0 and Landsberg erecta (Ler ecotypes.

  15. Short-term effect of single and combined exposures to nitrogen dioxide and ozone on DNA synthesis and cell proliferation in rat lungs

    International Nuclear Information System (INIS)

    Approximately three month old male Sprague-Dawley rats, free of specific pathogens, were exposed to either 1.8 ppm nitrogen dioxide (NO2) or 0.45 ppm ozone (O3) or to a combination of both of these gases. The time-course of oxidant exposure showed that for all exposure regimens, incorporation of tritated-thymidine into rat lung DNA peaked at forty-eight hours of continuous exposure and declined by seventy-two hours of exposure. The injury-repair curves for the O3 exposure and the combined exposure groups were similar. The labeling indexes (LI) for the terminal bronchioles, the alveolar type 2 cells, and all other alveolar interstitial cells at forty-eight hours of exposure demonstrated that the magnitude of alveolar epithelial injury was significantly greater when O3 was present. After forty-eight hours of exposure NO2 alone did not produce a measurable acute inflammatory response. The LI for the pulmonary alveolar macrophages suggests that there maybe in in-situ proliferation of these cells in response to oxidant exposure. There was a highly significant influx of neutrophils and an elevated protein content of cell-free lavage fluids indicative of an acute inflammatory response for the O3 exposed and the combined exposure groups

  16. Structural of the class II enzyme of human liver alcohol dehydrogenase: combined cDNA and protein sequence determination of the π subunit

    International Nuclear Information System (INIS)

    The class II enzyme of human liver alcohol dehydrogenase was isolated, carboxymethylated, and cleaved with CNBr and proteolytic enzymes. Sequence analysis of peptides established structures corresponding to the π subunit. Two segments from the C-terminal region unique to π were selected for synthesis of oligodeoxyribonucleotide probes to screen a human liver cDNA library constructed in plasmid pT4. Sequence analysis of two identical hybridization-positive clones with cDNA inserts of about 2000 nucleotides gave the entire coding region of the π subunit, a 61-nucleotide 5' noncoding region and a 741-nucleotide 3' noncoding region containing four possible polyadenylation sites. Translation of the coding region yields a 391-residue polypeptide, which in all regions except the C-terminal segment corresponds to the protein structure as determined directly by peptide analysis. With the class I numbering system, the exception concerns a residue exchange at position 368, the actual C-terminus which is Phe-374 by peptide data but a 12 residue extension by cDNA data, and possibly two further residue exchanges at positions 303 and 312. The size difference might indicate the existence of posttranslational modifications of the mature protein or, in combination with the residue exchanges, the existence of polymorphism at the locus for class II subunits. The π subunit analyzed directly results in a 379-residue polypeptide and is the only class II size thus far known to occur in the mature protein. Comparison of the π structure with those of the class I subunits (α, β, and γ) reveals a homology with extensive differences. Large variations in segments affecting relationships at the active site and the area of subunit interactions account for the significant alterations of enzymatic specificities and other properties that differentiate class II from class I enzymes

  17. Combination of treatment with death receptor 5-specific antibody with therapeutic HPV DNA vaccination generates enhanced therapeutic anti-tumor effects.

    Science.gov (United States)

    Tseng, Chih Wen; Monie, Archana; Trimble, Cornelia; Alvarez, Ronald D; Huh, Warner K; Buchsbaum, Donald J; Straughn, J Michael; Wang, Mei-Cheng; Yagita, Hideo; Hung, Chien-Fu; Wu, T-C

    2008-08-12

    There is currently a vital need for the development of novel therapeutic strategies for the control of advanced stage cancers. Antigen-specific immunotherapy and the employment of antibodies against the death receptor 5 (DR5) have emerged as two potentially promising strategies for cancer treatment. In the current study, we hypothesize that the combination of treatment with the anti-DR5 monoclonal antibody, MD5-1 with a DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 (HPV-16) E7 antigen (CRT/E7(detox)) administered via gene gun would lead to further enhancement of E7-specific immune responses as well as anti-tumor effects. Our results indicated that mice bearing the E7-expressing tumor, TC-1 treated with MD5-1 monoclonal antibody followed by CRT/E7(detox) DNA vaccination generated the most potent therapeutic anti-tumor effects as well as highest levels of E7-specific CD8+ T cells among all the groups tested. In addition, treatment with MD5-1 monoclonal antibody was capable of rendering the TC-1 tumor cells more susceptible to lysis by E7-specific cytotoxic T lymphocytes. Our findings serve as an important foundation for future clinical translation.

  18. Allelic frequency distributions of 21 non-combined DNA index system STR loci in a Russian ethnic minority group from Inner Mongolia, China*

    Science.gov (United States)

    Wang, Hong-dan; Shen, Chun-mei; Liu, Wen-juan; Zhang, Yu-dang; Yang, Guang; Yan, Jiang-wei; Qin, Hai-xia; Zhu, Bo-feng

    2013-01-01

    We studied the allelic frequency distributions and statistical forensic parameters of 21 new short tandem repeat (STR) loci and the amelogenin locus, which are not included in the combined DNA index system (CODIS), in a Russian ethnic minority group from the Inner Mongolia Autonomous Region, China. A total of 114 bloodstain samples from unrelated individuals were extracted and co-amplified with four fluorescence-labeled primers in a multiplex polymerase chain reaction (PCR) system. Using capillary electrophoresis, the PCR products of the 21 STR loci were separated and genotyped. A total of 161 alleles were observed in the Russian ethnic minority group, and corresponding allelic frequencies ranged from 0.0044 to 0.5965. The 21 non-CODIS STR loci of the Russian ethnic minority group were characterized by high genetic diversity and therefore may be useful for elucidating the population’s genetic background, for individual identification, and for paternity testing in forensic practice. PMID:23733431

  19. Allelic frequency distributions of 21 non-combined DNA index system STR loci in a Russian ethnic minority group from Inner Mongolia, China.

    Science.gov (United States)

    Wang, Hong-dan; Shen, Chun-mei; Liu, Wen-juan; Zhang, Yu-dang; Yang, Guang; Yan, Jiang-wei; Qin, Hai-xia; Zhu, Bo-feng

    2013-06-01

    We studied the allelic frequency distributions and statistical forensic parameters of 21 new short tandem repeat (STR) loci and the amelogenin locus, which are not included in the combined DNA index system (CODIS), in a Russian ethnic minority group from the Inner Mongolia Autonomous Region, China. A total of 114 bloodstain samples from unrelated individuals were extracted and co-amplified with four fluorescence-labeled primers in a multiplex polymerase chain reaction (PCR) system. Using capillary electrophoresis, the PCR products of the 21 STR loci were separated and genotyped. A total of 161 alleles were observed in the Russian ethnic minority group, and corresponding allelic frequencies ranged from 0.0044 to 0.5965. The 21 non-CODIS STR loci of the Russian ethnic minority group were characterized by high genetic diversity and therefore may be useful for elucidating the population's genetic background, for individual identification, and for paternity testing in forensic practice.

  20. Molecular Beacon Enables Combination of Highly Processive and Highly Sensitive Rolling Circle Amplification Readouts for Detection of DNA-Modifying Enzymes

    DEFF Research Database (Denmark)

    Kristoffersen, Emil Laust; Gonzales, Maria; Stougaard, Magnus;

    2015-01-01

    Here we present an optimized readout format for detection of the circularized products from a DNA-based sensor for measurement of DNA-modifying enzymes including DNA Topoisomerase I. The basic design of the DNA-sensor relies on the use of a substrate that can be circularized by the activity of DN...

  1. Triploblastic relationships with emphasis on the acoelomates and the position of Gnathostomulida, Cycliophora, Plathelminthes, and Chaetognatha: a combined approach of 18S rDNA sequences and morphology.

    Science.gov (United States)

    Giribet, G; Distel, D L; Polz, M; Sterrer, W; Wheeler, W C

    2000-09-01

    Triploblastic relationships were examined in the light of molecular and morphological evidence. Representatives for all triploblastic "phyla" (except Loricifera) were represented by both sources of phylogenetic data. The 18S ribosomal (rDNA) sequence data for 145 terminal taxa and 276 morphological characters coded for 36 supraspecific taxa were combined in a total evidence regime to determine the most consistent picture of triploblastic relationships for these data. Only triploblastic taxa are used to avoid rooting with distant outgroups, which seems to happen because of the extreme distance that separates diploblastic from triploblastic taxa according to the 18S rDNA data. Multiple phylogenetic analyses performed with variable analysis parameters yield largely inconsistent results for certain groups such as Chaetognatha, Acoela, and Nemertodermatida. A normalized incongruence length metric is used to assay the relative merit of the multiple analyses. The combined analysis having the least character incongruence yields the following scheme of relationships of four main clades: (1) Deuterostomia [((Echinodermata + Enteropneusta) (Cephalochordata (Urochordata + Vertebrata)))]; (2) Ecdysozoa [(((Priapulida + Kinorhyncha) (Nematoda + Nematomorpha)) ((Onychophora + Tardigrada) Arthropoda))]; (3) Trochozoa [((Phoronida + Brachiopoda) (Entoprocta (Nemertea (Sipuncula (Mollusca (Pogonophora (Echiura + Annelida)))))))]; and (4) Platyzoa [((Gnathostomulida (Cycliophora + Syndermata)) (Gastrotricha + Plathelminthes))]. Chaetognatha, Nemertodermatida, and Bryozoa cannot be assigned to any one of these four groups. For the first time, a data analysis recognizes a clade of acoelomates, the Platyzoa (sensu Cavalier-Smith, Biol. Rev. 73:203-266, 1998). Other relationships that corroborate some morphological analyses are the existence of a clade that groups Gnathostomulida + Syndermata (= Gnathifera), which is expanded to include the enigmatic phylum Cycliophora, as sister group

  2. Phylogenetic relationships of Middle American cichlids (Cichlidae, Heroini) based on combined evidence from nuclear genes, mtDNA, and morphology.

    Science.gov (United States)

    Rícan, Oldrich; Zardoya, Rafael; Doadrio, Ignacio

    2008-12-01

    Heroine cichlids are the second largest and very diverse tribe of Neotropical cichlids, and the only cichlid group that inhabits Mesoamerica. The taxonomy of heroines is complex because monophyly of most genera has never been demonstrated, and many species groups are without applicable generic names after their removal from the catch-all genus Cichlasoma (sensu Regan, 1905). Hence, a robust phylogeny for the group is largely wanting. A rather complete heroine phylogeny based on cytb sequence data is available [Concheiro Pérez, G.A., Rícan O., Ortí G., Bermingham, E., Doadrio, I., Zardoya, R. 2007. Phylogeny and biogeography of 91 species of heroine cichlids (Teleostei: Cichlidae) based on sequences of the cytochrome b gene. Mol. Phylogenet. Evol. 43, 91-110], and in the present study, we have added and analyzed independent data sets (nuclear and morphological) to further confirm and strengthen the cytb-phylogenetic hypothesis. We have analyzed a combined cytb-nuclear (RAG1 and two S7 introns) data set of 48 species representing main heroine lineages to achieve further resolution of heroine higher taxonomic levels and a combined cytb-morphological data set of 92 species to stabilize generic taxonomy. The recovered phylogenies supported the circumamazonian--CAM--Heroini (sensu Concheiro Peréz et al., 2007) as a monophyletic group, that could be divided into six main clades: (1) australoheroines (the southernmost heroine genus Australoheros), (2) nandopsines (the Antillean genus Nandopsis), (3) caquetaines (including the north western Amazonian genera Caquetaia and Heroina), (4) astatheroines (including Astatheros, Herotilapia and Rocio), (5) amphilophines (including Amphilophus and related genera), and (6) herichthyines (including Herichthyis and related genera). Nuclear and mitochondrial data partitions arrived at highly congruent topologies. Suprageneric relationships were influenced mainly by the nuclear signal, as well as the most basal phylogenetic position

  3. Predicted sub-populations in a marine shrimp proteome as revealed by combined EST and cDNA data from multiple Penaeus species

    Directory of Open Access Journals (Sweden)

    Kotewong Rattanawadee

    2010-11-01

    Full Text Available Abstract Background Many species of marine shrimp in the Family Penaeidae, viz. Penaeus (Litopenaeus vannamei, Penaeus monodon, Penaeus (Fenneropenaeus chinensis, and Penaeus (Marsupenaeus japonicus, are animals of economic importance in the aquaculture industry. Yet information about their DNA and protein sequences is lacking. In order to predict their collective proteome, we combined over 270,000 available EST and cDNA sequences from the 4 shrimp species with all protein sequences of Drosophila melanogaster and Caenorhabditis elegans. EST data from 4 other crustaceans, the crab Carcinus maenas, the lobster Homarus americanus (Decapoda, the water flea Daphnia pulex, and the brine shrimp Artemia franciscana were also used. Findings Similarity searches from EST collections of the 4 shrimp species matched 64% of the protein sequences of the fruit fly, but only 45% of nematode proteins, indicating that the shrimp proteome content is more similar to that of an insect than a nematode. Combined results with 4 additional non-shrimp crustaceans increased matching to 78% of fruit fly and 56% of nematode proteins, suggesting that present shrimp EST collections still lack sequences for many conserved crustacean proteins. Analysis of matching data revealed the presence of 4 EST groups from shrimp, namely sequences for proteins that are both fruit fly-like and nematode-like, fruit fly-like only, nematode-like only, and non-matching. Gene ontology profiles of proteins for the 3 matching EST groups were analyzed. For non-matching ESTs, a small fraction matched protein sequences from other species in the UniProt database, including other crustacean-specific proteins. Conclusions Shrimp ESTs indicated that the shrimp proteome is comprised of sub-populations of proteins similar to those common to both insect and nematode models, those present specifically in either model, or neither. Combining small EST collections from related species to compensate for their

  4. Establishment and Identification of Chinese Hamster Ovary Cell Lines with Stable Expression of Soluble CD40 Ligands

    Directory of Open Access Journals (Sweden)

    JIANG Hua-wei

    2014-09-01

    Full Text Available Objective: To establish the Chinese Hamster Ovary (CHO cell lines with stable expression of soluble CD40 ligands (sCD40L. Methods: Recombinant plasmid pIRES2-EGFP-sCD40L, enzyme digestion and sequencing identification were obtained by cloning sCD40L coding sequences into eukaryotic expression vector pIRES2-EGFP from carrier pDC316-sCD40 containing sCD40L. CHO cells were transfected by electroporation, followed by screening of resistant clones with G418, after which monoclones were obtained by limited dilution assay and multiply cultured. Flow cytometer and reverted fluorescence microscope were applied to observe the expression of green fluorescent protein, while sCD40L expression was detected by polymerase chain reaction (PCR, reverse transcription-polymerase chain reaction (RT-PCR and enzyme-linked immunosorbent assay (ELISA from aspects of deoxyribose nucleic acid (DNA, messenger ribonucleic acid (mRNA and protein, respectively. CHO-sCD40L was cultured together with MDA-MB-231 cells to compare the expression changes of surface molecule fatty acid synthase (Fas by flow cytometer and observe the apoptosis of MDA-MB-231 cells after Fas activated antibodies (CH-11 were added 24 h later. Results: Plasmid pIRES2-EGFP-sCD40L was successfully established, and cell lines with stable expression of sCD40L were obtained with cloned culture after CHO cell transfection, which was named as B11. Flow cytometer and reverted fluorescence microscope showed >90% expression of green fluorescent protein, while PCR, RT-PCR and ELISA suggested integration of sCD40L genes into cell genome DNA, transcription of sCD40L mRNA and sCD40L protein expression being (4.5±2.1 ng/mL in the supernatant of cell culture, respectively. After co-culture of B11 and MDA-MB-231 cells, the surface Fas expression of MDA-MB-231 cells was increased from (3±1.02 % to (34.8±8.75%, while the apoptosis rate 24 h after addition of CH11 from (5.4±1.32% to (20.7±5.24%, and the differences

  5. Redox-responsive targeted gelatin nanoparticles for delivery of combination wt-p53 expressing plasmid DNA and gemcitabine in the treatment of pancreatic cancer

    International Nuclear Information System (INIS)

    Pancreatic adenocarcinoma is one of the most dreaded cancers with very low survival rate and poor prognosis to the existing frontline chemotherapeutic drugs. Gene therapy in combination with a cytotoxic agent could be a promising approach to circumvent the limitations of previously attempted therapeutic interventions. We have developed a redox-responsive thiolated gelatin based nanoparticle system that efficiently delivers its payload in the presence of glutathione-mediated reducing intra-cellular environment and could be successfully used for site-specific wt-p53 expressing plasmid DNA as well as gemcitabine delivery by targeting epidermal growth factor receptor (EGFR). Efficacy studies were performed in subcutaneous human adenocarcinoma bearing SCID beige mice along with molecular level p53 plasmid and apoptotic marker expression by PCR and western blot for all study groups. Efficacy studies demonstrate an improved in vivo targeting efficiency resulting in increased transfection efficiency and tumor growth suppression. In all the treatment groups, the targeted nanoparticles showed better anti-tumor activity than their non-targeted as well as non-encapsulated, naked therapeutic agent counterparts (50.1, 61.7 and 77.3% tumor regression by p53 plasmid alone, gemcitabine alone and in combination respectively). Molecular analysis revealed a higher mRNA expression of transfected p53 gene, its corresponding protein and that the tumor cell death in all treatment groups was due to the induction of apoptotic pathways. Gene/drug combination treatment significantly improves the therapeutic performance of the delivery system compared to the gene or drug alone treated groups. Anti-tumor activity of the thiolated gelatin loaded wt-p53 plasmid or gemcitabine-based therapy was attributed to their ability to induce cell apoptosis, which was confirmed by a marked increase in mRNA level of proapoptotic transcription factors, as well as, protein apoptotic biomarker expression and

  6. Construction of recombinant eukaryotic expression plasmid containing murine CD40 ligand gene and its expression in H22 cells

    Institute of Scientific and Technical Information of China (English)

    Yong-Fang Jiang; Yan He; Guo-Zhong Gong; Jun Chen; Chun-Yan Yang; Yun Xu

    2005-01-01

    AIM: To construct a recombinant murine CD40 ligand (mCD40L) eukaryotic expression vector for gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: mCD40L cDNA was synthesized by RT-PCR with the specific primers and directly cloned into T vector to generate middle recombinant. After digestion with restriction endonuclease, the target fragment was subcloned into the multi-clone sites of the eukaryotic vector. The constructed vector was verified by enzyme digestion and sequencing,and the product expressed was detected by RT-PCR and immunofluorescence methods.RESULTS: The full-length mCD40L-cDNA was successfully cloned into the eukaryotic vector through electrophoresis,and mCD40L gene was integrated into the genome of infected H22 cells by RT-PCR. Murine CD40L antigen molecule was observed in the plasma of mCD40L-H22 by indirect immuno-fluorescence staining.CONCLUSION: The recombined mCD40L eukaryotic expression vector can be expressed in H22 cell line. It providesexperimental data for gene therapy and target therapy ofhepatocellular carcinoma.

  7. Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes

    Science.gov (United States)

    Arunrut, Narong; Kampeera, Jantana; Sirithammajak, Sarawut; Sanguanrut, Piyachat; Proespraiwong, Porranee; Suebsing, Rungkarn; Kiatpathomchai, Wansika

    2016-01-01

    Acute hepatopancreatic necrosis disease (AHPND) is a component cause of early mortality syndrome (EMS) of shrimp. In 2013, the causative agent was found to be unique isolates of Vibrio parahaemolyticus (VPAHPND) that contained a 69 kbp plasmid (pAP1) carrying binary Pir-like toxin genes PirvpA and PirvpB. In Thailand, AHPND was first recognized in 2012, prior to knowledge of the causative agent, and it subsequently led to a precipitous drop in shrimp production. After VPAHPND was characterized, a major focus of the AHPND control strategy was to monitor broodstock shrimp and post larvae for freedom from VPAHPND by nucleic acid amplification methods, most of which required use of expensive and sophisticated equipment not readily available in a shrimp farm setting. Here, we describe a simpler but equally sensitive approach for detection of VPAHPND based on loop-mediated isothermal amplification (LAMP) combined with unaided visual reading of positive amplification products using a DNA-functionalized, ssDNA-labled nanogold probe (AuNP). The target for the special set of six LAMP primers used was the VPAHPND PirvpA gene. The LAMP reaction was carried out at 65°C for 45 min followed by addition of the red AuNP solution and further incubation at 65°C for 5 min, allowing any PirvpA gene amplicons present to hybridize with the probe. Hybridization protected the AuNP against aggregation, so that the solution color remained red upon subsequent salt addition (positive test result) while unprotected AuNP aggregated and underwent a color change from red to blue and eventually precipitated (negative result). The total assay time was approximately 50 min. The detection limit (100 CFU) was comparable to that of other commonly-used methods for nested PCR detection of VPAHPND and 100-times more sensitive than 1-step PCR detection methods (104 CFU) that used amplicon detection by electrophoresis or spectrophotometry. There was no cross reaction with DNA templates derived from non

  8. Superior Protective Immunity against Murine Listeriosis by Combined Vaccination with CpG DNA and Recombinant Salmonella enterica Serovar Typhimurium▿ †

    Science.gov (United States)

    Berchtold, Christina; Panthel, Klaus; Jellbauer, Stefan; Köhn, Brigitte; Roider, Elisabeth; Partilla, Miriam; Heesemann, Jürgen; Endres, Stefan; Bourquin, Carole; Rüssmann, Holger

    2009-01-01

    Preexisting antivector immunity can severely compromise the ability of Salmonella enterica serovar Typhimurium live vaccines to induce protective CD8 T-cell frequencies after type III secretion system-mediated heterologous protein translocation in orally immunized mice. To circumvent this problem, we injected CpG DNA admixed to the immunodominant p60217-225 peptide from Listeria monocytogenes subcutaneously into BALB/c mice and coadministered a p60-translocating Salmonella strain by the orogastric route. The distribution of tetramer-positive p60217-225-specific effector and memory CD8 T cells was analyzed by costaining of lymphocytes with CD62L and CD127. In contrast to the single oral application of recombinant Salmonella or single immunization with CpG and p60, in the spleens from mice immunized with a combination of both vaccine types a significantly higher level of p60-specific CD8 T cells with a predominance of the effector memory T-cell subset was detected. In vivo protection studies revealed that this CD8 T-cell population conferred sterile protective immunity against a lethal infection with L. monocytogenes. However, p60-specific central memory CD8 T cells induced by single vaccination with CpG and p60 were not able confer effective protection against rapidly replicating intracellular Listeria. In conclusion, we provide compelling evidence that the combination of Salmonella type III-mediated antigen delivery and CpG immunization is an attractive novel vaccination strategy to modulate CD8 differentiation patterns toward distinct antigen-specific T-cell subsets with favorable protective capacities. PMID:19797070

  9. Deciphering transcriptional networks that govern Coffea arabica seed development using combined cDNA array and real-time RT-PCR approaches.

    Science.gov (United States)

    Salmona, Jordi; Dussert, Stéphane; Descroix, Frédéric; de Kochko, Alexandre; Bertrand, Benoît; Joët, Thierry

    2008-01-01

    Due to its economic importance, Coffea arabica is becoming the subject of increasing genomic research and, in particular, the genes involved in the final chemical composition of the bean and the sensorial quality of the coffee beverage. The aim of the present study was to decipher the transcriptional networks that govern the development of the C. arabica seed, a model for non-orthodox albuminous seeds of tropical origin. For this purpose, we developed a transcriptomic approach combining two techniques: targeted cDNA arrays, containing 266 selected candidate gene sequences, and real-time RT-PCR on a large subset of 111 genes. The combination of the two techniques allowed us to limit detection of false positives and to reveal the advantages of using large real-time RT-PCR screening. Multivariate analysis was conducted on both datasets and results were broadly convergent. First, principle component analysis (PCA) revealed a dramatic re-programming of the transcriptional machinery between early cell division and elongation, storage and maturation phases. Second, hierarchical clustering analysis (HCA) led to the identification of 11 distinct patterns of gene expression during seed development as well as to the detection of genes expressed at specific developmental stages that can be used as functional markers of phenological changes. In addition, this study led to the description of gene expression profiles for quality-related genes, most of them formerly uncharacterised in Coffea. Their involvement in storage compound synthesis and accumulation during endosperm development and final metabolic re-adjustments during maturation is discussed. PMID:18026845

  10. Molecular cloning, sequencing, and expression analysis of cDNA encoding metalloprotein II (MP II) induced by single and combined metals (Cu(II), Cd(II)) in polychaeta Perinereis aibuhitensis.

    Science.gov (United States)

    Yang, Dazuo; Zhou, Yibing; Zhao, Huan; Zhou, Xiaoxiao; Sun, Na; Wang, Bin; Yuan, Xiutang

    2012-11-01

    We amplified and analyzed the complete cDNA of metalloprotein II (MP II) from the somatic muscle of the polychaete Perinereis aibuhitensis, the full length cDNA is 904 bp encoding 119 amino acids. The MP II cDNA sequence was subjected to BLAST searching in NCBI and was found to share high homology with hemerythrin of other worms. MP II expression of P. aibuhitensis exposed to single and combined metals (Cu(II), Cd(II)) was analyzed using real time-PCR. MP II mRNA expression increased at the start of Cu(II) exposure, then decreased and finally return to the normal level. Expression pattern of MP II under Cd(II) exposure was time- and dose-dependent. MP II expression induced by a combination of Cd(II) and Cu(II) was similar to that induced by Cd(II) alone.

  11. Allelic frequency distributions of 21 non-combined DNA index system STR loci in a Russian ethnic minority group from Inner Mongolia, China

    Institute of Scientific and Technical Information of China (English)

    Hong-dan WANG; Chun-mei SHEN; Wen-juan LIU; Yu-dang ZHANG; Guang YANG; Jiang-wei YAN; Hai-xia QIN

    2013-01-01

    We studied the allelic frequency distributions and statistical forensic parameters of 21 new short tandem repeat (STR) loci and the amelogenin locus,which are not included in the combined DNA index system (CODIS),in a Russian ethnic minority group from the Inner Mongolia Autonomous Region,China.A total of 114 bloodstain samples from unrelated individuals were extracted and co-amplified with four fluorescence-labeled primers in a multiplex polymerase chain reaction (PCR) system.Using capillary electrophoresis,the PCR products of the 21 STR loci were separated and genotyped.A total of 161 alleles were observed in the Russian ethnic minority group,and corresponding allelic frequencies ranged from 0.0044 to 0.5965.The 21 non-CODIS STR loci of the Russian ethnic minority group were characterized by high genetic diversity and therefore may be useful for elucidating the population's genetic background,for individual identification,and for paternity testing in forensic practice.

  12. Allele frequencies of combined DNA index system (CODIS) and non-CODIS short tandem repeat loci in Goiás, Central Brazil.

    Science.gov (United States)

    Rodovalho, R G; Santos, G S; Cavalcanti, L M; Moura, B F S M; Rodrigues, E L; Lima, P R; Gigonzac, M A D; Vieira, T C

    2015-01-01

    In studies of human identification, obtaining a high standard of outcomes and satisfactory conclusions are directly related to the use of highly polymorphic molecular markers. In addition to the combined DNA index system (CODIS) group, it is also important to implement non-CODIS markers into the analysis, as they increase the power of discrimination. During the identification process, it is essential to consider the genetic variation among distinct groups of populations, as the allele frequencies are directly associated with the power of discrimination. However, the population of Goiás, a State located in Central Brazil, is characterized by a highly mixed population due to its diverse ethnic origins. In this study, a survey of the allelic frequencies in the Goiás population was carried out using a molecular assembly composed of 21 autosomal loci both from and external to the CODIS group. The new data, for some of the markers used, were statistically similar to those from previous studies. This consistency means that the use of these markers might serve as a parameter for future population comparisons. The results from these analyses further our knowledge of the study of human identification.

  13. Metformin synergizes 5-fluorouracil, epirubicin, and cyclophosphamide (FEC) combination therapy through impairing intracellular ATP production and DNA repair in breast cancer stem cells.

    Science.gov (United States)

    Soo, Jaslyn Sian-Siu; Ng, Char-Hong; Tan, Si Hoey; Malik, Rozita Abdul; Teh, Yew-Ching; Tan, Boon-Shing; Ho, Gwo-Fuang; See, Mee-Hoong; Taib, Nur Aishah Mohd; Yip, Cheng-Har; Chung, Felicia Fei-Lei; Hii, Ling-Wei; Teo, Soo-Hwang; Leong, Chee-Onn

    2015-10-01

    Metformin, an AMPK activator, has been reported to improve pathological response to chemotherapy in diabetic breast cancer patients. To date, its mechanism of action in cancer, especially in cancer stem cells (CSCs) have not been fully elucidated. In this study, we demonstrated that metformin, but not other AMPK activators (e.g. AICAR and A-769662), synergizes 5-fluouracil, epirubicin, and cyclophosphamide (FEC) combination chemotherapy in non-stem breast cancer cells and breast cancer stem cells. We show that this occurs through an AMPK-dependent mechanism in parental breast cancer cell lines. In contrast, the synergistic effects of metformin and FEC occurred in an AMPK-independent mechanism in breast CSCs. Further analyses revealed that metformin accelerated glucose consumption and lactate production more severely in the breast CSCs but the production of intracellular ATP was severely hampered, leading to a severe energy crisis and impairs the ability of CSCs to repair FEC-induced DNA damage. Indeed, addition of extracellular ATP completely abrogated the synergistic effects of metformin on FEC sensitivity in breast CSCs. In conclusion, our results suggest that metformin synergizes FEC sensitivity through distinct mechanism in parental breast cancer cell lines and CSCs, thus providing further evidence for the clinical relevance of metformin for the treatment of cancers. PMID:26276035

  14. Induction of Immune Tolerance in Asthmatic Mice by Vaccination with DNA Encoding an Allergen–Cytotoxic T Lymphocyte-Associated Antigen 4 Combination

    OpenAIRE

    Zhang, Fang; Huang, Gang; Hu, Bo; Song, Yong; Shi, Yi

    2011-01-01

    Allergen-specific immunotherapy is a potential treatment for allergic diseases. We constructed an allergen–cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)-encoding DNA vaccine, administered it directly to antigen-presenting cells (APCs), and investigated its ability and mechanisms to ameliorate allergic airway inflammation in an asthmatic mouse model. An allergen-CTLA-4 DNA plasmid (OVA-CTLA-4-pcDNA3.1) encoding an ovalbumin (OVA) and the mouse CTLA-4 extracellular domain was constructed...

  15. Ancient DNA

    DEFF Research Database (Denmark)

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair......ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair...

  16. Fish oil in combination with high or low intakes of linoleic acid lowers plasma triacylglycerols but does not affect other cardiovascular risk markers in healthy men

    DEFF Research Database (Denmark)

    Damsgaard, Camilla T.; Frøkiær, Hanne; Andersen, Anders D.;

    2008-01-01

    with a high- or low-LA intake affects overall CVD risk profile. Healthy men (n = 64) were randomized to 5 mL/d fish oil capsules (FO) [mean intake 3.1 g/d (n-3) LCPUFA] or olive oil capsules (control) and to oils and spreads with either a high (S/B) or a low (R/K) LA content, resulting in a 7.3 g/d higher LA......Both (n-3) long-chain PUFA (LCPUFA) and linoleic acid [LA, 18:2(n-6)] improve cardiovascular disease (CVD) risk factors, but a high-LA intake may weaken the effect of (n-3) LCPUFA. In a controlled, double-blind, 2 x 2-factorial 8-wk intervention, we investigated whether fish oil combined......, C-reactive protein, interleukin-6, vascular cell adhesion molecule-1, P-selectin, oxidized LDL, cluster of differentiation antigen 40 ligand (CD40L), adiponectin, or fasting or postprandial BP or HR after adjustment for body weight changes. In conclusion, neither fish oil supplementation nor the LA...

  17. Identification of human DNA in forensic evidence by loop-mediated isothermal amplification combined with a colorimetric gold nanoparticle hybridization probe.

    Science.gov (United States)

    Watthanapanpituck, Khanistha; Kiatpathomchai, Wansika; Chu, Eric; Panvisavas, Nathinee

    2014-11-01

    A DNA test based on loop-mediated isothermal amplification (LAMP) and colorimetric gold nanoparticle (AuNP) hybridization probe to detect the presence of human DNA in forensic evidence was developed. The LAMP primer set targeted eight regions of the human cytochrome b, and its specificity was verified against the DNA of 11 animal species, which included animals closely related to humans, such as chimpanzee and orangutan. By using the AuNP probe, sequence-specific LAMP product could be detected and the test result could be visualized through the change in color. The limit of detection was demonstrated with reproducibility to be as low as 718 fg of genomic DNA, which is equivalent to approximately 100 plasmid DNA copies containing the cytochrome b DNA target region. A simple DNA extraction method for the commonly found forensic biological samples was also devised to streamline the test process. This LAMP-AuNP human DNA test showed to be a robust, specific, and cost-effective tool for the forensic identification of human specimens without requiring sophisticated laboratory instruments.

  18. Identification of human DNA in forensic evidence by loop-mediated isothermal amplification combined with a colorimetric gold nanoparticle hybridization probe.

    Science.gov (United States)

    Watthanapanpituck, Khanistha; Kiatpathomchai, Wansika; Chu, Eric; Panvisavas, Nathinee

    2014-11-01

    A DNA test based on loop-mediated isothermal amplification (LAMP) and colorimetric gold nanoparticle (AuNP) hybridization probe to detect the presence of human DNA in forensic evidence was developed. The LAMP primer set targeted eight regions of the human cytochrome b, and its specificity was verified against the DNA of 11 animal species, which included animals closely related to humans, such as chimpanzee and orangutan. By using the AuNP probe, sequence-specific LAMP product could be detected and the test result could be visualized through the change in color. The limit of detection was demonstrated with reproducibility to be as low as 718 fg of genomic DNA, which is equivalent to approximately 100 plasmid DNA copies containing the cytochrome b DNA target region. A simple DNA extraction method for the commonly found forensic biological samples was also devised to streamline the test process. This LAMP-AuNP human DNA test showed to be a robust, specific, and cost-effective tool for the forensic identification of human specimens without requiring sophisticated laboratory instruments. PMID:24827529

  19. Combined effects of DNA methyltransferase 1 and 3A polymorphisms and urinary total arsenic levels on the risk for clear cell renal cell carcinoma.

    Science.gov (United States)

    Yang, Shu-Mei; Huang, Chao-Yuan; Shiue, Horng-Sheng; Pu, Yeong-Shiau; Hsieh, Yi-Hsun; Chen, Wei-Jen; Lin, Ying-Chin; Hsueh, Yu-Mei

    2016-08-15

    Our previous study showed that high urinary total arsenic levels were associated with higher odds ratio (OR) for renal cell carcinoma (RCC). Single nucleotide polymorphisms (SNPs) of DNA methyltransferases (DNMTs) might influence DNMT enzyme activity associated with tumorigenesis. In this study, we investigated the association of five SNPs from DNMT1 (rs8101626 and rs2228611), DNMT3A (rs34048824 and rs1550117), and DNMT3B (rs1569686) with the risk of clear cell renal cell carcinoma (ccRCC). We also examined the combined effects of DNMT genotypes and urinary arsenic levels on ccRCC risk. We conducted a hospital-based case-control study, which included 293 subjects with ccRCC and 293 age- and gender-matched controls. The urinary arsenic species were determined by a high performance liquid chromatography-linked hydride generator and atomic absorption spectrometry. Genotypes were investigated using polymerase chain reaction and restriction fragment length polymorphism analyses. We observed that the DNMT1 rs8101626 G/G genotype was significantly associated with reduced odds ratio (OR) of ccRCC [OR=0.38, 95% confidence interval (CI) 0.14-0.99]. Subjects with concurrent DNMT1 rs8101626 A/A+A/G and DNMT3A rs34048824 T/T+T/C genotypes had significantly higher OR for ccRCC [OR=2.88, 95% CI 1.44-5.77]. Participants with the high-risk genotype of DNMT1 rs8101626 and DNMT3A rs34048824 with concurrently high urinary total arsenic levels had even higher OR of ccRCC in a dose-response manner. This is the first study to evaluate variant DNMT1 rs8101626 and DNMT3A rs34048824 genotypes that modify the arsenic-related ccRCC risk in a geographic area without significant arsenic exposure in Taiwan. PMID:27292127

  20. Combining pseudo dinucleotide composition with the Z curve method to improve the accuracy of predicting DNA elements: a case study in recombination spots.

    Science.gov (United States)

    Dong, Chuan; Yuan, Ya-Zhou; Zhang, Fa-Zhan; Hua, Hong-Li; Ye, Yuan-Nong; Labena, Abraham Alemayehu; Lin, Hao; Chen, Wei; Guo, Feng-Biao

    2016-08-16

    Pseudo dinucleotide composition (PseDNC) and Z curve showed excellent performance in the classification issues of nucleotide sequences in bioinformatics. Inspired by the principle of Z curve theory, we improved PseDNC to give the phase-specific PseDNC (psPseDNC). In this study, we used the prediction of recombination spots as a case to illustrate the capability of psPseDNC and also PseDNC fused with Z curve theory based on a novel machine learning method named large margin distribution machine (LDM). We verified that combining the two widely used approaches could generate better performance compared to only using PseDNC with a support vector machine based (SVM-based) model. The best Mathew's correlation coefficient (MCC) achieved by our LDM-based model was 0.7037 through the rigorous jackknife test and improved by ∼6.6%, ∼3.2%, and ∼2.4% compared with three previous studies. Similarly, the accuracy was improved by 3.2% compared with our previous iRSpot-PseDNC web server through an independent data test. These results demonstrate that the joint use of PseDNC and Z curve enhances performance and can extract more information from a biological sequence. To facilitate research in this area, we constructed a user-friendly web server for predicting hot/cold spots, HcsPredictor, which can be freely accessed from . In summary, we provided a united algorithm by integrating Z curve with PseDNC. We hope this united algorithm could be extended to other classification issues in DNA elements. PMID:27410247

  1. Combining pseudo dinucleotide composition with the Z curve method to improve the accuracy of predicting DNA elements: a case study in recombination spots.

    Science.gov (United States)

    Dong, Chuan; Yuan, Ya-Zhou; Zhang, Fa-Zhan; Hua, Hong-Li; Ye, Yuan-Nong; Labena, Abraham Alemayehu; Lin, Hao; Chen, Wei; Guo, Feng-Biao

    2016-08-16

    Pseudo dinucleotide composition (PseDNC) and Z curve showed excellent performance in the classification issues of nucleotide sequences in bioinformatics. Inspired by the principle of Z curve theory, we improved PseDNC to give the phase-specific PseDNC (psPseDNC). In this study, we used the prediction of recombination spots as a case to illustrate the capability of psPseDNC and also PseDNC fused with Z curve theory based on a novel machine learning method named large margin distribution machine (LDM). We verified that combining the two widely used approaches could generate better performance compared to only using PseDNC with a support vector machine based (SVM-based) model. The best Mathew's correlation coefficient (MCC) achieved by our LDM-based model was 0.7037 through the rigorous jackknife test and improved by ∼6.6%, ∼3.2%, and ∼2.4% compared with three previous studies. Similarly, the accuracy was improved by 3.2% compared with our previous iRSpot-PseDNC web server through an independent data test. These results demonstrate that the joint use of PseDNC and Z curve enhances performance and can extract more information from a biological sequence. To facilitate research in this area, we constructed a user-friendly web server for predicting hot/cold spots, HcsPredictor, which can be freely accessed from . In summary, we provided a united algorithm by integrating Z curve with PseDNC. We hope this united algorithm could be extended to other classification issues in DNA elements.

  2. Combining combing and secondary ion mass spectrometry to study DNA on chips using 13C and 15N labeling [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Armelle Cabin-Flaman

    2016-06-01

    Full Text Available Dynamic secondary ion mass spectrometry (D-SIMS imaging of combed DNA – the combing, imaging by SIMS or CIS method – has been developed previously using a standard NanoSIMS 50 to reveal, on the 50 nm scale, individual DNA fibers labeled with different, non-radioactive isotopes in vivo and to quantify these isotopes. This makes CIS especially suitable for determining the times, places and rates of DNA synthesis as well as the detection of the fine-scale re-arrangements of DNA and of molecules associated with combed DNA fibers. Here, we show how CIS may be extended to 13C-labeling via the detection and quantification of the 13C14N- recombinant ion and the use of the 13C:12C ratio, we discuss how CIS might permit three successive labels, and we suggest ideas that might be explored using CIS.

  3. 基于PSCA的肽-DNA复合疫苗的构建及其免疫学效应的检测%Construction of a PSCA based peptide-DNA combined vaccine and its CTLs killing activity identification

    Institute of Scientific and Technical Information of China (English)

    刘勇; 靳凤烁; 张克勤

    2011-01-01

    This study is aimed to construct a PSCA based peptide-DNA combined vaccine and identify its CTLs killing activity. PLL-PSCA14-22 was obtained by coupling the positively charged poly-lysine (PLL) with a CTL epitope peptide PSCA 14-22. Then DNA/peptide combined vaccine was formed by the self-assembly of a cationic peptide PLL-PSCA14-22 with a plasmid encoding human full-length PSCA gene through electrostatic interactions. The preparation of this combined vaccine was then identified with both DNA retardation and Dnase I protection experiments. The transfection efficiency and CTLs killing activity were evaluated by RT-PCR and LDH release assay, respectively. The protection of DNA was begun at the charge ratios of peptide/DNA≥2, at which DNA was completely retarded in the DNA retardation assay. The PSCA transcripts was existed in the combined vaccine-transfected Hela cells and CTLs killing activity was induced in vivo, which led to the obvious killing of PSCA-positive LNCaP cells. The PSCA-based peptide-DNA combined vaccine can be transfected into eukaryotic cells and induce specific CTLs response and kill PSCA-positive LNCaP cells effectively, which may pave a way for further studies in animals.%目的 设计、制备和鉴定PLL-PSCA 14-22/-pcDNA3.1 (+)-PSCA复合疫苗及其免疫学效应的检测.方法 将带正电荷的多聚赖氨酸(PLL)通过化学方法与PSCA的一个CTL表位肽PSCA14-22偶联,将上述阳离子多肽PLL-PSCA 14-22与带负电荷的编码PSCA的重组质粒pcDNA3.1 (+)-PSCA利用正负电荷相吸作用制备靶向PSCA的肽-DNA复合疫苗;通过DNA阻滞实验和DNase Ⅰ保护实验鉴定制备的疫苗;将制备成功的复合疫苗体外转染Hela细胞,通过RT-PCR检测该疫苗的转染效率;并进一步通过LDH释放实验检测该疫苗对PSCA阳性的前列腺癌细胞系LNCaP的特异性杀伤效应.结果 当PLL-PSCA 14-22多肽与pcDNA3.1 (+)-PSCA重组质粒正负电荷比≥2时,DNA电泳条带完全消失,即重组质粒pcDNA

  4. OCA-B integrates B cell antigen receptor-, CD40L- and IL 4-mediated signals for the germinal center pathway of B cell development.

    OpenAIRE

    Qin, X F; Reichlin, A; Luo, Y.; Roeder, R. G.; Nussenzweig, M.C.

    1998-01-01

    Many of the key decisions in lymphocyte differentiation and activation are dependent on integration of antigen receptor and co-receptor signals. Although there is significant understanding of these receptors and their signaling pathways, little is known about the molecular requirements for signal integration at the level of activation of gene expression. Here we show that in primary B cells, expression of the B-cell specific transcription coactivator OCA-B (also known as OBF-1 or Bob-1) is re...

  5. Application of cpDNA Marker Technique in Parents Selection and Combination of Potato Breeding%cpDNA标记在马铃薯杂交亲本选配中的应用

    Institute of Scientific and Technical Information of China (English)

    胡祚; 郝大海; 段晓艳; 王兆富; 李周; 苏跃; 冯泽蔚; 李灿辉

    2011-01-01

    Parents selection and combination is the initiate and important step to potato breeding program. U-sing chloroplast DNA ( cpDNA) marker technique, the author detected cytoplast genetic background of the parents and 380 clones derived from hybrid true potato seeds of two cross combinations (Shepody x CIP 004 n = 187 and PB 06 x CIP 004 n = 193 ) . Results showed that Shepody harbored T-type markers of cpDNA which was subjected to the Tuberosum subspecies of Solarium tuberosum L. , but both PB 06 and CIP 004 had A-type markers of cpDNA which belonged to the Andigena subspecies of Solarium tuberosum L.. All clones tested could also be divided into two groups which separately shared the same cpDNA markers with female parents, and the averaged growth period of each group tended to female parents, respectively. The aboved results demonstrated that there was a great potential for cpDNA marker technique in parents selection and combination of potato breeding program.%亲本选配是马铃薯杂交育种工作的重点和难点.利用叶绿体DNA(cpDNA)标记方法,对两个马铃薯杂交组合(Shepody×CIP 004和PB 06×CIP 004)的亲本、及其杂交实生种子后代群体的细胞质遗传背景进行了分析.结果显示:Shepody拥有T-型cpDNA标记,属于普通栽培亚种(Solanium tuberosum ssp.tuberosum);CIP004和PB06拥有A-型cpDNA标记,均属于安第斯栽培亚种(Solanium tuberosum ssp.andigena);在cpDNA两个标记位点(H1和H2)上,2个杂交组合实生种子后代群体的细胞质遗传背景分别与其母本完全一致,平均生育期分别与其母本接近.上述结果表明,cpDNA标记技术在马铃薯杂交亲本选配和早世代选育中具有重要的应用价值.

  6. Modeling and time-dependent dynamics of processes of stimulated depolymerization, auto-repairing, degradation and radiation curing of DNA macromolecules and biopolymers at separated and combined actions of ionizing irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Vysotskii, Vladimir I. E-mail: viv@vhome.kiev.ua; Pinchuk, Anatoliy O.; Kornilova, Alla A.; Samoylenko, Igor I

    2001-12-01

    The time-dependent dynamics of the formation, relaxation and auto-repairing of double breaks of DNA macromolecules at the combined radiation action and non-radiation processes of degradation (e.g. by free radicals) were considered. The auto-repairing of DNA double breaks is connected with the peculiarities of long-range interaction of nucleotide charges, atoms and molecules in the intracellular milieu. The properties of intracellular liquid and the characteristics of force interaction between the end-pairs of nucleotides in the area of DNA break in response to radiation are changed. Each kind of radiation is characterized by a certain effectiveness of the double DNA break formation but simultaneously one creates the conditions for their liquidation. On the basis of the analysis and correlation of these processes the time-dependent theory for DNA degradation was created, including hormesis phenomenon, radiation antagonism, the validity of anomaly influence of low and large doses at sharp and chronic radiation and other effects. The qualitative and quantitative correspondences of the theory and experimental results of radiation biology were obtained.

  7. A randomised, phase II trial of the DNA-hypomethylating agent 5-aza-2′-deoxycytidine (decitabine) in combination with carboplatin vs carboplatin alone in patients with recurrent, partially platinum-sensitive ovarian cancer

    Science.gov (United States)

    Glasspool, R M; Brown, R; Gore, M E; Rustin, G J S; McNeish, I A; Wilson, R H; Pledge, S; Paul, J; Mackean, M; Hall, G D; Gabra, H; Halford, S E R; Walker, J; Appleton, K; Ullah, R; Kaye, S

    2014-01-01

    Background: Our previous laboratory and clinical data suggested that one mechanism underlying the development of platinum resistance in ovarian cancer is the acquisition of DNA methylation. We therefore tested the hypothesis that the DNA hypomethylating agent 5-aza-2′-deoxycytodine (decitabine) can reverse resistance to carboplatin in women with relapsed ovarian cancer. Methods: Patients progressing 6–12 months after previous platinum therapy were randomised to decitabine on day 1 and carboplatin (AUC 6) on day 8, every 28 days or carboplatin alone. The primary objective was response rate in patients with methylated hMLH1 tumour DNA in plasma. Results: After a pre-defined interim analysis, the study closed due to lack of efficacy and poor treatment deliverability in 15 patients treated with the combination. Responses by GCIG criteria were 9 out of 14 vs 3 out of 15 and by RECIST were 6 out of 13 vs 1 out of 12 for carboplatin and carboplatin/decitabine, respectively. Grade 3/4 neutropenia was more common with the combination (60% vs 15.4%) as was G2/3 carboplatin hypersensitivity (47% vs 21%). Conclusions: With this schedule, the addition of decitabine appears to reduce rather than increase the efficacy of carboplatin in partially platinum-sensitive ovarian cancer and is difficult to deliver. Patient-selection strategies, different schedules and other demethylating agents should be considered in future combination studies. PMID:24642620

  8. Aberrant DNA Methylation of P16, MGMT, and hMLH1 Genes in Combination with MTHFR C677T Genetic Polymorphism in gastric cancer

    OpenAIRE

    Song, Binbin; Ai, Jiang; Kong, Xianghong; Liu, Dexin; Li, Jun

    2013-01-01

    Objective: We aimed to explore the association of P16, MGMT and HMLH1 with gastric cancer and their relation with Methylenetetrahydrofolate reductase (MTHFR). Methods: 322 gastric patients who were confirmed with pathological diagnosis were included in our study. Aberrant DNA methylation of P16, MGMT and HMLH1 and polymorphisms of MTHFR C677T and A1298C were detected using PCR-RFLP. Results: The proportions of DNA hypermethylation in P16, MGMT and hMLH1 genes in gastric cancer tissues were 75...

  9. Solid-phase based on-chip DNA purification through a valve-free stepwise injection of multiple reagents employing centrifugal force combined with a hydrophobic capillary barrier pressure.

    Science.gov (United States)

    Zhang, Hainan; Tran, Hong Hanh; Chung, Bong Hyun; Lee, Nae Yoon

    2013-03-21

    In this paper, we demonstrate a simple technique for sequentially introducing multiple sample liquids into microchannels driven by centrifugal force combined with a hydrophobic barrier pressure and apply the technique for performing solid-phase based on-chip DNA purification. Three microchannels with varying widths, all equipped with independent sample reservoirs at the inlets, were fabricated on a hydrophobic elastomer, poly(dimethylsiloxane) (PDMS). First, glass beads were packed inside the reaction chamber, and a whole cell containing the DNA extract was introduced into the widest channel by applying centrifugal force for physical adsorption of the DNA onto the glass beads. Next, washing and elution solutions were sequentially introduced into the intermediate and narrowest microchannels, respectively, by gradually increasing the amount of centrifugal force. Through a precise manipulation of the centrifugal force, the DNA adsorbed onto the glass beads was successfully washed and eluted in a continuous manner without the need to introduce each solution manually. A stepwise injection of liquids was successfully demonstrated using multiple ink solutions, the results of which corresponded well with the theoretical analyses. As a practical application, the D1S80 locus of human genomic DNA, which is widely used for forensic purposes, was successfully purified using the microdevice introduced in this study, as demonstrated through successful target amplification. This will pave the way for the construction of a control-free valve system for realizing on-chip DNA purification, which is one of the most labor-intensive and hard-to-miniaturize components, on a greatly simplified and miniaturized platform employing hydrophobic PDMS.

  10. Treatment of brain glioblastoma multiforme with pcDNA3.1-Egr. 1p-p16 combined with gamma knife radiation: An experimental study on nude mice

    Directory of Open Access Journals (Sweden)

    Liu Wenke

    2013-01-01

    Full Text Available Background: High post-operative recurrence and poor prognosis are likely to be related to the infiltrative growth of the glioblastoma multiforme (GBM. Objectives: The primary objective of this study is to investigate the possible synergistic effect of the combined treatment of gamma knife radio-surgery (GKRS and gene therapy for GBM and secondary objective is to explore the role of GKRS for the temporal and spatial regulation of the gene expression. Materials and Methods: The study performed on 70 nude mice and randomly divided into seven groups. Subcutaneous injection of human GBM tumor cells (T98G was carried out to establish the animal models. Various doses of liposome-mediated pcDNA3.1-Egr. 1p-p16 recombinant plasmid were transfected through intra-tumor injection. GKRS was scheduled following the plasmid transfection. Tumor volumes were measured every 4 days after the treatment. Subcutaneous tumor nodule specimens were collected to analyze the cell apoptosis and p16 gene expression using terminal-deoxynucleoitidyl transferase mediated nick end labeling staining and reverse transcription-polymerase chain reaction. Tumor volumes, levels of cell apoptosis and p16 gene expression were compared between groups. Results: Rates of tumor growth were significantly lower in the pcDNA3.1-Egr. 1p-p16 plasmid + GKRS groups than that in the remaining groups 28 days following the GKRS management. The p16mRNA expression was noted in both of the pcDNA3.1-Egr. 1p-p16 plasmid group and the pcDNA3.1-Egr. 1p-p16 plasmid + GKRS with marginal-dose of 20 Gy group. The level of messenger ribonucleic acid expression was higher in the pcDNA3.1-Egr. 1p-p16 plasmid + GKRS with the marginal-dose of 20 Gy group, with a markedly increased apoptotic and necrotic cells, than that in the pcDNA3.1-Egr. 1p-p16 plasmid group. Conclusions: In animal studies, pcDNA3.1-Egr. 1p-p16 in combination with GKRS is a preferable management option for the GBM to the sole use of GKRS or gene

  11. Aberrant DNA Methylation of P16, MGMT, and hMLH1 Genes in Combination with MTHFR C677T Genetic Polymorphism in gastric cancer

    Science.gov (United States)

    Song, Binbin; Ai, Jiang; Kong, Xianghong; Liu, Dexin; Li, Jun

    2013-01-01

    Objective: We aimed to explore the association of P16, MGMT and HMLH1 with gastric cancer and their relation with Methylenetetrahydrofolate reductase (MTHFR). Methods: 322 gastric patients who were confirmed with pathological diagnosis were included in our study. Aberrant DNA methylation of P16, MGMT and HMLH1 and polymorphisms of MTHFR C677T and A1298C were detected using PCR-RFLP. Results: The proportions of DNA hypermethylation in P16, MGMT and hMLH1 genes in gastric cancer tissues were 75.2% (242/322), 27.6% (89/322) and 5.3% (17/322), respectively. In the remote normal-appearing tissues, 29.5% (95/322) and 16.1%(52/322) showed hypermethylation in P16 and MGMT genes, respectively. We found a significantly higher proportion of DNA hypermethylation of P16 in patients with N1 TNM stage in cancer tissues and remote normal-appearing tissues (P<0.05). Similarly, we found DNA hypermethylation of MGMT had significantly higher proportion in N1 and M1 TNM stage (P<0.05). Individuals with homozygotes (TT) of MTHFR C677T had significant risk of DNA hypermethylation of MGMT in cancer tissues [OR (95% CI)=4.27(1.76-7.84)], and a significant risk was also found in those carrying MTHFR 677CT/TT genotype [OR (95% CI)= 3.27(1.21-4.77)]. Conclusion: We found the aberrant hypermethylation of cancer-related genes, such as P16, MGMT and HMLH1, could be predictive biomarkers for detection of gastric cancer. PMID:24550949

  12. Forensic DNA profiling and database.

    Science.gov (United States)

    Panneerchelvam, S; Norazmi, M N

    2003-07-01

    The incredible power of DNA technology as an identification tool had brought a tremendous change in crimnal justice . DNA data base is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. This article discusses the essential steps in compilation of COmbined DNA Index System (CODIS) on validated polymerase chain amplified STRs and their use in crime detection.

  13. Forensic DNA Profiling and Database

    OpenAIRE

    Panneerchelvam, S.; Norazmi, M. N.

    2003-01-01

    The incredible power of DNA technology as an identification tool had brought a tremendous change in crimnal justice . DNA data base is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. This article discusses the essential steps in compilation of COmbined DNA Index System (CODIS) on validated polymerase chain amplified STRs and their use in crime detection.

  14. Innovations. DNA detectives.

    OpenAIRE

    May, M

    1999-01-01

    To understand the many potential causes and resulting consequences of DNA damage, scientists first need methods to detect it. Canadian scientists X. Chris Le and Michael Weinfeld, with help from U.S. molecular biologist Steven Leadon, developed a selective, sensitive technique for measuring DNA damage. The scientists combined a thymine glycol antibody with thymine glycol to selectively tag a specific type of DNA damage. They then added a second antibody with fluorescing properties, and used l...

  15. Identification and characterization of new DNA G-quadruplex binders selected by a combination of ligand and structure-based virtual screening approaches.

    Science.gov (United States)

    Alcaro, Stefano; Musetti, Caterina; Distinto, Simona; Casatti, Margherita; Zagotto, Giuseppe; Artese, Anna; Parrotta, Lucia; Moraca, Federica; Costa, Giosuè; Ortuso, Francesco; Maccioni, Elias; Sissi, Claudia

    2013-02-14

    Nowadays, it has been demonstrated that DNA G-quadruplex arrangements are involved in cellular aging and cancer, thus boosting the discovery of selective binders for these DNA secondary structures. By taking advantage of available structural and biological information on these structures, we performed a high throughput in silico screening of commercially available molecules databases by merging ligand- and structure-based approaches by means of docking experiments. Compounds selected by the virtual screening procedure were then tested for their ability to interact with the human telomeric G-quadruplex folding by circular dichroism, fluorescence spectroscopy, and photodynamic techniques. Interestingly, our screening succeeded in retrieving a new promising scaffold for G-quadruplex binders characterized by a psoralen moiety. PMID:23294188

  16. Simultaneous RNA-DNA FISH.

    Science.gov (United States)

    Lai, Lan-Tian; Meng, Zhenyu; Shao, Fangwei; Zhang, Li-Feng

    2016-01-01

    A highly useful tool for studying lncRNAs is simultaneous RNA-DNA FISH, which reveals the localization and quantitative information of RNA and DNA in cellular contexts. However, a simple combination of RNA FISH and DNA FISH often generates disappointing results because the fragile RNA signals are often damaged by the harsh conditions used in DNA FISH for denaturing the DNA. Here, we describe a robust and simple RNA-DNA FISH protocol, in which amino-labeled nucleic acid probes are used for RNA FISH. The method is suitable to detect single-RNA molecules simultaneously with DNA.

  17. The variety identification of QUINA in Brazil based on a combined analysis of DNA barcoding-origin-morphology%基于 DNA 条形码-产地-形态分析联用的巴西马钱属植物药 QUINA 基原鉴定研究

    Institute of Scientific and Technical Information of China (English)

    臧艺玫; 顾选; 余春霞; 肖瑶; 赵百孝; 刘春生; 马长华; 韩丽

    2015-01-01

    目的:通过 DNA 条形码—产地—形态分析联用的方法对巴西马钱属植物药 QUINA进行基原鉴定研究,为这种药材的开发利用提供依据。方法通过提取样品 DNA、PCR 扩增及双向测序,获得样品的内转录间隔区(internal transcribed spacer,ITS)序列,利用 NCBI 数据库中的 BLAST功能计算相似度,对其基原进行 DNA 条形码鉴定;根据其产地信息对 DNA 条形码鉴定结果进行筛选和分析;依据其形态特征验证鉴定结果。结果本次收集的植物类药材 QUINA 来源于马钱科马钱属植物 Strychnos pseudo quina A. St.-Hil.的根皮。结论 DNA 条形码—产地—形态三者联用的方法可弥补单一鉴定方法的不足,能够快速、准确的对未知样品进行基原鉴定。%Objective To study the variety identification of QUINA of Strychnos Linn. with the combined analysis of DNA barcoding-origin-morphology and provide an evidence for the exploitation of this kind of herbal in Brazil. Methods ITS sequences were amplified by PCR from genomic DNA and sequenced. , BLAST( basic local alignment search tool) in NCBI database was used to calculate the similarity of sequences to identify the variety of samples; then the DNA barcoding identification results were screened and analyzed in reference to its origin information; finally, the morphological characteristics of samples was used to verify the accuracy of identification. Results QUINA collected from market in Brazil was the root bark of Strychnos pseudo quina A. St. -Hil. Conclusion The combined analysis of DNA barcoding-origin-morphology could make up for the deficit of onefold identification method. It contributes to identifying the variety of unidentified samples quickly and accurately.

  18. Cleaving DNA with DNA

    Science.gov (United States)

    Carmi, Nir; Balkhi, Shameelah R.; Breaker, Ronald R.

    1998-03-01

    A DNA structure is described that can cleave single-stranded DNA oligonucleotides in the presence of ionic copper. This ``deoxyribozyme'' can self-cleave or can operate as a bimolecular complex that simultaneously makes use of duplex and triplex interactions to bind and cleave separate DNA substrates. Bimolecular deoxyribozyme-mediated strand scission proceeds with a kobs of 0.2 min-1, whereas the corresponding uncatalyzed reaction could not be detected. The duplex and triplex recognition domains can be altered, making possible the targeted cleavage of single-stranded DNAs with different nucleotide sequences. Several small synthetic DNAs were made to function as simple ``restriction enzymes'' for the site-specific cleavage of single-stranded DNA.

  19. Induction of immune tolerance in asthmatic mice by vaccination with DNA encoding an allergen-cytotoxic T lymphocyte-associated antigen 4 combination.

    Science.gov (United States)

    Zhang, Fang; Huang, Gang; Hu, Bo; Song, Yong; Shi, Yi

    2011-05-01

    Allergen-specific immunotherapy is a potential treatment for allergic diseases. We constructed an allergen-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)-encoding DNA vaccine, administered it directly to antigen-presenting cells (APCs), and investigated its ability and mechanisms to ameliorate allergic airway inflammation in an asthmatic mouse model. An allergen-CTLA-4 DNA plasmid (OVA-CTLA-4-pcDNA₃.₁) encoding an ovalbumin (OVA) and the mouse CTLA-4 extracellular domain was constructed and transfected into COS-7 cells to obtain the fusion protein OVA-CTLA-4, which was able to bind the B7 ligand on dendritic cells (DCs), and induced CD25⁺ Foxp3⁺ regulatory T (Treg) cells by the coculture of naive CD4⁺ T cells with DCs in vitro. In an animal study, BALB/c mice were sensitized and challenged with OVA to establish the asthmatic model. Vaccination with a high dose of OVA-CTLA-4-pcDNA₃.₁ significantly decreased interleukin-4 (IL-4) and IL-5 levels and eosinophil counts and prevented OVA-induced reduction of the gamma interferon level in the bronchoalveolar lavage fluid. In addition, these mice suffered less severe airway inflammation and had lower levels of OVA-specific IgE and IgG1 titers in serum. Also, high-dose OVA-CTLA-4-pcDNA₃.₁ vaccination inhibited the development of airway hyperreactivity and prevented OVA-induced reduction of the percentages of Foxp3⁺ Treg cells in the spleen. Our results indicate that a high dose of allergen-CTLA-4-encoding DNA vaccine was more effective in preventing an allergen-induced Th2-skewed immune response through the induction of Treg cells and may be a new alternative therapy for asthma. PMID:21346053

  20. A combined approach of DNA probe and RFLP for family and species identification of larval stages of commercially important aquatic species: A study on the surfclam Spisula solidissima

    Digital Repository Service at National Institute of Oceanography (India)

    Achuthankutty, C.T.

    - tively charged nylon membrane (Boehri- nger Mannheim, Germany), 1 ?l of the heated PCR product was applied and the DNA was crosslinked to the membrane by UV light (1200 mV). The Genius Non- radioactive Nucleic Acid Labeling and Detection System was used... probe in dot-blot hybridization. The probe hybridized with 12 PCR products from larvae and controls of only S. solidissima and M. lateralis and not with other bivalve controls and negative con- trol (Fig. 2A). The membrane was later exposed...

  1. A tumor targeted therapy study of PcDNA-sTRAIL combined with 131I-angiostatin on lewis lung garcinoma nude mice%131I-血管抑素联合PcDNA-sTRAIL在荷瘤小鼠模型肿瘤靶向治疗的研究

    Institute of Scientific and Technical Information of China (English)

    张弦; 田琼; 魏龙晓; 周润锁; 徐海峰; 袁梦辉

    2012-01-01

    Objective :To build PcDNA-sTRAIL eukaryolic expression vector and amplify, purify it. Then observe the joint sntitumor effect of the PcDNA-sTRAIL combined with '" I -AS in Lewis Lung Carcinoma nude mice Methods ■ Through the PCR amplification and directional cloning technology to build PcDN A-sTR AIL eukaryotic express carrier, me '" 1 to mark AS. The mark rate could reach 85 'A and exhibit good in vitro stability. After successfully constructed a tumor-burdened nude mice model, then we got a tumor-burdened mice SPECT imaging. Next, we completed a tumor targeted therapy study of restructuring PcON A-sTRAIL combined with '" I -AS. Result the overage volume of tumor in the PcDNA-sTRAIL and '" I-AS combined treatment group is smaller the other single treated groups. Conclusion ?Thi* research firstly suggested the combined treatment of inhibiting tumor angiogenesis effect, promoting the tumor cell apoptosis effect, and radiation treatment to fight cancer. We expect we could not only take full advantages of the three methods, but also provide powerful experimental evidences for combination treatment of radionuclide and the immune therapy.%目的:构建PcDNA-sTRAIL真核表达载体并进行扩增、纯化,采用131I标记血管抑素(AS)并完成了PcDNA-sTRAIL与131I-AS在荷瘤裸鼠体内的联合抑瘤试验.方法;通过PCR扩增和定向克隆技术成功构建PcDNA-sTRAIL真核表达载体,采用131I标记血管抑素,标记率达85%且体外稳定性好,成功构建了荷瘤裸鼠模型,并进行了荷瘤小鼠联合治疗后SPECT显像和病理结果,完成PcDNA-sTRAIL与131I-AS在荷瘤裸鼠体内的联合抑瘤试验.结果:荷瘤小鼠PcDNA-sTRAIL与131I-AS联合用药治疗期间肿瘤平均体积较其他单独治疗组增长缓慢.结论:本研究将抑制肿瘤血管生成、促肿瘤细胞凋亡和放射性核素的内照射三重作用相结合并用于抗肿瘤治疗,期望不仅能充分发挥三者的优势,又能为核素内照射与免疫

  2. Detection of Eleven Pathogenic Bacterium Using DNA Microarray Combined With Multiplex PCR%多重PCR结合基因芯片技术检测11种致病菌方法的建立

    Institute of Scientific and Technical Information of China (English)

    贺晨; 孙鸿燕; 邵丽筠; 刘金华; 张春秀; 刘娅

    2011-01-01

    Objective To establish a rapid ,accurate detection method for 11 kinds of pathogenic bacteria using DNA microarray combined with multiplex PCR .Methods The special genes of these Bacterium including Shigella, Listeria monocytogenes ,Escherichi-a coli O157, Staphylococcus aureus , Bacillus cereus , Campylobacter jejuni, Proteusbacillus vulgaris , C . perfringens , Vibiro para-haemolyticus ,Salmonella typhi ,Salmonella enteritidis were as target genes for multiplex PCR respectively ,then primers and captured oligonucleotide probes were designed and synthesized .After multiplex PCR system and hybridization reaction were optimized ,the multr-plex PCR products were hybridizes with DNA microarray ,which contained specific probes of eleven pathogenic bacterium .Scanner was used to determinant the types of bacterium .Results The DNA microarray assay can detect 11 kinds of pathogenic bacterium specially .The sensitivity of the DNA may reach 2?0-3 ng .Conclusion To detect 11 kinds of pathogenic bacteria using DNA microarray combined with multiplex PCR is specific ,sensiu've ,practical.%目的 建立一种运用多重PCR方法结合基因芯片技术快速、准确检测11种常见致病菌的方法.方法 筛选志贺氏菌、肠炎沙门氏菌、伤寒沙门氏菌、大肠杆菌O157、副溶血性弧菌、普通变形杆菌、蜡样芽孢杆菌、金黄色葡萄球菌、单核细胞增生李斯特菌、产气荚膜梭菌、空肠弯曲菌的特异基因作为目的 基因.设计相应的引物及探针,进行多重PCR扩增,制备寡核苷酸芯片.将多重PCR扩增产物与带有11种特异探针的基因芯片杂交.用扫描仪扫描,判定细菌种类.结果 该基因芯片可特异性地检测11种致病菌,具有良好的特异性,基因组DNA检测灵敏度可达2×10-3ng.结论 多重PCR结合基因芯片技术检测11种不同致病菌的方法特异性好,灵敏度高,具有较好的实用性.

  3. DNA与结核抗体联合检测对结核性胸膜炎的诊断意义%Significance of combined detection of DNA and tuberculosis antibody in the diagnosis of tuberculous pleurisy

    Institute of Scientific and Technical Information of China (English)

    李玉明; 郭明日; 张立

    2014-01-01

    目的:探讨DNA和结核抗体(TB-Ab)联合检测在结核性胸膜炎的临床诊断意义。方法分别采用斑点金免疫渗滤法和实时荧光定量PCR法对95例结核性胸腔积液患者及45例非结核性胸腔积液患者进行TB-Ab和结核分枝杆菌DNA检测,并对结果进行分析比较。结果结核性胸膜炎组结核分枝杆菌 DNA 检测阳性率为56.8%,显著高于非结核性胸膜炎组(4.4%),差异具有统计学意义(P<0.05)。结核性胸膜炎组TB-Ab检测阳性率为58.9%,显著高于非结核性胸膜炎组(6.6%),差异具有统计学意义(P<0.05)。两项指标应用结核性胸膜炎组阳性率可提高为78.9%,显著高于涂片镜检以及结核菌培养单项检测阳性率,差异具有统计学意(P<0.01)。结论结核分枝杆菌DNA 与TB-Ab 联合检测可明显提高结核性胸膜炎的阳性检出率,而且快速、简便、易操作。%Objective To disscuss the significance of combined detection of DNA and tuberculosis antibody(TB-Ab)in the diag-nosis of tuberculous pleurisy.Methods Dot immunogold filtration assay and real-time fluorescence PCR method were respectively used to detect TB-Ab and DNA of 95 cases of tuberculous pleural effusion patients(tuberculosis pleurisy group)and 45 cases of pa-tients without tuberculous pleural effusion(non-tuberculous pleurisy group).Results were analyzed and compared.Results The positive rate of Mycobacterium tuberculosis DNA in tuberculosis pleurisy group was 5 6 .8%,which was higher than that of non-tu-berculous pleurisy group(4.4%),with significant difference between them(P<0.05).The positive rate of TB-Ab in tuberculosis pleurisy group was 58.9%,higher than that of non tuberculous pleurisy group (6.6%),and the difference was statistically signifi-cant (P<0.05).Combined application of DNA and TB-Ab,the positive rate of tuberculosis pleurisy group could be increased to 78.9%,significantly higher

  4. Comparative investigations of sodium arsenite, arsenic trioxide and cadmium sulphate in combination with gamma-radiation on apoptosis, micronuclei induction and DNA damage in a human lymphoblastoid cell line

    International Nuclear Information System (INIS)

    In the field of radiation protection the combined exposure to radiation and other toxic agents is recognised as an important research area. To elucidate the basic mechanisms of simultaneous exposure, the interaction of the carcinogens and environmental toxicants cadmium and two arsenic compounds, arsenite and arsenic trioxide, in combination with gamma-radiation in human lymphoblastoid cells (TK6) were investigated. Gamma-radiation induced significant genotoxic effects such as micronuclei formation, DNA damage and apoptosis, whereas arsenic and cadmium had no significant effect on these indicators of cellular damage at non-toxic concentrations. However, in combination with gamma-radiation arsenic trioxide induced a more than additive apoptotic rate compared to the sum of the single effects. Here, the level of apoptotic cells was increased, in a dose-dependent way, up to two-fold compared to the irradiated control cells. Arsenite did not induce a significant additive effect at any of the concentrations or radiation doses tested. On the other hand, arsenic trioxide was less effective than arsenite in the induction of DNA protein cross-links. These data indicate that the two arsenic compounds interact through different pathways in the cell. Cadmium sulphate, like arsenite, had no significant effect on apoptosis in combination with gamma-radiation at low concentrations and, at high concentrations, even reduced the radiation-induced apoptosis. An additive effect on micronuclei induction was observed with 1 μM cadmium sulphate with an increase of up to 80% compared to the irradiated control cells. Toxic concentrations of cadmium and arsenic trioxide seemed to reduce micronuclei induction. The results presented here indicate that relatively low concentrations of arsenic and cadmium, close to those occurring in nature, may interfere with radiation effects. Differences in action of the two arsenic compounds were identified

  5. DNA media storage

    Institute of Scientific and Technical Information of China (English)

    Christy M.Bogard; Eric C.Rouchka; Benjamin Arazi

    2008-01-01

    In 1994. University of Southern California computer scientist,Dr.Leonard Adleman solved the Hamiltonian path problem using DNA as a computational mechanism.He proved the principle that DNA computing could be used to solve computationally complex problems.Because of the limitations in discovery time,resource requirements,and sequence mismatches,DNA computing has not yet become a commonly accepted practice.However,advancements are continually being discovered that are evolving the field of DNA computing.Practical applications of DNA are not restricted to computation alone.This research presents a novel approach in which DNA could be used as a means of storing files.Through the use of multiple sequence alignment combined with intelligent heuristics,the most probabilistic file contents can be determined with minimal errors.

  6. Improved Immunogenicity of a Vaccination Regimen Combining a DNA Vaccine Encoding Brucella melitensis Outer Membrane Protein 31 (Omp31) and Recombinant Omp31 Boosting▿

    Science.gov (United States)

    Cassataro, Juliana; Velikovsky, Carlos A.; Bruno, Laura; Estein, Silvia M.; de la Barrera, Silvia; Bowden, Raúl; Fossati, Carlos A.; Giambartolomei, Guillermo H.

    2007-01-01

    In the present study, we report an attempt to improve the immunogenicity of the Omp31 antigen by a DNA prime-protein boost immunization regimen. We immunized BALB/c mice with an Omp31 DNA vaccine (pCIOmp31) followed by boosting with recombinant Omp31 (rOmp31) in incomplete Freund's adjuvant and characterized the resulting immune responses and the protective efficacy against Brucella ovis and B. melitensis infection. Immunoglobulin G1 (IgG1) and IgG2a titers were higher in sera from pCIOmp31/rOmp31-immunized mice than in sera from mice immunized with pCIOmp31 or rOmp31 alone. Splenocytes from pCIOmp31/rOmp31-immunized mice produced significantly higher levels of gamma interferon than did those from mice given rOmp31 alone. In contrast, interleukin 2 (IL-2) production levels were comparable between the two groups of immunized mice. Cells from all immunized mice produced undetectable levels of IL-4. Notably, rOmp31 stimulated IL-10 production in the pCIOmp31/rOmp31-immunized group but not in the pCIOmp31- or rOmp31-immunized group. Although the prime-boost regimen induced specific cytotoxic responses, these responses could not reach the levels achieved by the pCIOmp31 immunization. In conclusion, pCIOmp31 priming followed by rOmp31 boosting led to moderately improved protection against a challenge with B. ovis or B. melitensis. PMID:17428946

  7. 基于性状-显微-DNA 条形码联用的巴西牛至属植物药 HORTELA 基原鉴定研究%The variety identification of HORTELA in Brazil based on a combined analysis of macroscopic-mi-croscopic-DNA barcoding

    Institute of Scientific and Technical Information of China (English)

    余春霞; 臧艺玫; 顾选; 吴浩忠; 马长华; 韩丽; 刘春生; 赵百孝

    2015-01-01

    Objective To study the variety identification of HORTELA of Origanum Linn. in Brazil with a combined analysis of macroscopic-microscopic-DNA barcoding. Methods Macroscopic iden-tification was used to identify the authenticity of samples preliminarily; then the results was verified by mi-croscopic identification; finally, ITS sequences were amplified by PCR and sequenced, and BLAST in NCBI ( basic local alignment search tool) database was used to calculate the similarity of sequences to identify variety of samples. Results The combined analysis of macroscopic-microscopic-DNA barcoding indicated that Hortela collected from market in Brazil is the dried overground part of Origanum elongatum (Bonnet) Emb. & Maire. Conclusion HORTELA may be a multi-source herb ,which originates from the dried overground part of Origanum elongatum(Bonnet) Emb. & Maire and Mentha rotundifolia (Linn. ) Huds.%目的:利用性状—显微—DNA 条形码联用的方法对巴西牛至属植物药 HORTELA 进行基原鉴定研究。方法应用性状鉴定的方法初步对药材进行真伪鉴别;再应用显微鉴定的方法验证性状鉴定结果;最后提取样品 DNA,扩增 ITS 序列,利用 NCBI 数据库中的 BLAST 功能分析相似度,对样品品种进行 DNA 条形码鉴定。结果性状—显微—DNA 条形码综合分析结果表明,该样品为牛至属植物 Origanum elongatum(Bonnet) Emb.& Maire.的干燥地上部分。结论 HORTELA 可能为多基原植物类药材,其来源为牛至属 Origanum elongatum (Bonnet) Emb.& Maire.和薄荷属Mentha rotundifolia (Linn.) Huds.干燥地上部分。

  8. X-linked Hyper IgM (HIGM1 in an African kindred: the first report from South Africa

    Directory of Open Access Journals (Sweden)

    Henderson Howard E

    2003-11-01

    Full Text Available Abstract Background The objective of this study was to describe the clinical and molecular features of the first South African family with X-linked hyper-IgM syndrome (HIGM1. Methods Diagnoses were based on immunoglobulin results and the absence of CD40 ligand (CD40L expression on activated T-cells. Complete molecular characterisation involved CD40L cDNA sequencing, and genomic DNA analysis by polymerase chain reaction amplification, restriction enzyme digestion and sequencing. A PCR-based diagnostic assay was established for carrier detection and prenatal diagnosis in this family. Results There were originally six children, three males and three females. The eldest boy died after being diagnosed with hypogammaglobulinaemia, before HIGM1 was considered. This disorder was diagnosed in the second eldest boy at the age of 5 years, after failing to detect CD40L expression on his activated T-cells. A deficiency of CD40L was also confirmed in the youngest male at the age of 5 years. Both younger brothers have since died of infections relating to HIGM1. Molecular investigation showed that exon 3 was deleted from the CD40L mRNA of the affected males. Genomic DNA analysis identified a 1.5 kilobase deletion, spanning exon 3 and including extended flanking intronic sequence. Carrier status in the mother was confirmed by RT-PCR of her CD40L mRNA. Genetic analysis of the three female children was deferred because they were below the legal consenting age of 18 years. A PCR-based assay for genomic DNA was established for easy identification of female carriers and affected males in the future. Conclusions This study confirmed the diagnosis of HIGM1 in the first South African family to be investigated and identified a novel mutation in the CD40L gene.

  9. CD40 ligand induced cytotoxicity in carcinoma cells is enhanced by inhibition of metalloproteinase cleavage and delivery via a conditionally-replicating adenovirus

    Directory of Open Access Journals (Sweden)

    Young Lawrence S

    2010-03-01

    Full Text Available Abstract Background CD40 and its ligand (CD40L play a critical role in co-ordinating immune responses. CD40 is also expressed in lymphoid malignancies and a number of carcinomas. In carcinoma cells the physiological outcome of CD40 ligation depends on the level of receptor engagement with low levels promoting cell survival and high levels inducing cell death. The most profound induction of cell death in carcinoma cells is induced by membrane-bound rather than recombinant soluble CD40L, but like other TNF family ligands, it is cleaved from the membrane by matrix metalloproteinases. Results We have generated a replication-deficient adenovirus expressing a mutant CD40L that is resistant to metalloproteinase cleavage such that ligand expression is retained at the cell membrane. Here we show that the mutated, cleavage-resistant form of CD40L is a more potent inducer of apoptosis than wild-type ligand in CD40-positive carcinoma cell lines. Since transgene expression via replication-deficient adenovirus vectors in vivo is low, we have also engineered a conditionally replicating E1A-CR2 deleted adenovirus to express mutant CD40L, resulting in significant amplification of ligand expression and consequent enhancement of its therapeutic effect. Conclusions Combined with numerous studies demonstrating its immunotherapeutic potential, these data provide a strong rationale for the exploitation of the CD40-CD40L pathway for the treatment of solid tumours.

  10. The variety identification of FARINHA DE LARANJA powder in Brazil based on a combined analysis of microscopic-DNA barcoding-origin%基于显微-DNA 条形码-产地分析联用的巴西柑橘属植物药 FARINHA DE LARANJA(枳壳)基原鉴定研究

    Institute of Scientific and Technical Information of China (English)

    臧艺玫; 马条华; 顾选; 余春霞; 王娟; 韩丽; 吴浩忠; 肖瑶; 赵百孝; 刘春生

    2015-01-01

    Objective o study the variety identification of a kind of herbal powder of FARINHA DE LARANJA of Citrus L. in Brazil with a combaned analysis of microscope - DNA barcoding - origin. Methods Microscopic identification was used to identify the variety of samples preliminarily; then ITS se-quences were amplified by PCR and sequenced, and BLAST in NCBI database was used to calculate the similarity of sequences to identify variety of samples. , Finally, the DNA barcoding identification results were screened and analyzed according to its geographical origin information. Results The combined analysis of microscopic - DNA barcoding - origin indicated that FARINHA DE LARANJA collected from market in Brazil was the dried fruit powder of Citrus aurantium. Conclusion The identification of Brazil FARINHA DE LARANJA could enrich the alternative resources of Zhiqiao and Zhishi.%目的:利用显微-DNA 条形码-产地分析联用的方法对巴西柑橘属植物药 FARINHA DE LARANJA 粉末进行基原鉴定研究。方法应用显微鉴定的方法对药材粉末的基原进行初步判定;再提取样品 DNA,扩增 ITS 序列,利用 NCBI 数据库中的 BLAST 功能分析相似度,对样品品种进行DNA 条形码鉴定;最后结合产地信息对数据库中的序列进行筛选。结果显微-DNA 条形码-产地综合分析结果表明,该样品为芸香科柑橘属 Citrus L.植物酸橙 Citrus aurantium 的干燥未成熟果实粉末。结论巴西植物药 FARINHA DE LARANJA 的鉴定可为扩大枳实、枳壳等中药材的药用资源提供依据。

  11. Combined low initial DNA damage and high radiation-induced apoptosis confers clinical resistance to long-term toxicity in breast cancer patients treated with high-dose radiotherapy

    Directory of Open Access Journals (Sweden)

    Bordón Elisa

    2011-06-01

    Full Text Available Abstract Background Either higher levels of initial DNA damage or lower levels of radiation-induced apoptosis in peripheral blood lymphocytes have been associated to increased risk for develop late radiation-induced toxicity. It has been recently published that these two predictive tests are inversely related. The aim of the present study was to investigate the combined role of both tests in relation to clinical radiation-induced toxicity in a set of breast cancer patients treated with high dose hyperfractionated radical radiotherapy. Methods Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma treated with high-dose hyperfractioned radical radiotherapy. Acute and late cutaneous and subcutaneous toxicity was evaluated using the Radiation Therapy Oncology Group morbidity scoring schema. The mean follow-up of survivors (n = 13 was 197.23 months. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp. Radiation-induced apoptosis (RIA at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. Results Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46. Radiation-induced apoptosis increased with radiation dose (median 12.36, 17.79 and 24.83 for 1, 2, and 8 Gy respectively. We observed that those "expected resistant patients" (DSB values lower than 1.78 DSB/Gy per 200 Mbp and RIA values over 9.58, 14.40 or 24.83 for 1, 2 and 8 Gy respectively were at low risk of suffer severe subcutaneous late toxicity (HR 0.223, 95%CI 0.073-0.678, P = 0.008; HR 0.206, 95%CI 0.063-0.677, P = 0.009; HR 0.239, 95%CI 0.062-0.929, P = 0.039, for RIA at 1, 2 and 8 Gy respectively in multivariate analysis. Conclusions A radiation-resistant profile is proposed, where those patients who presented lower levels of initial DNA damage and higher levels of radiation induced apoptosis were at low

  12. A combined de novo protein sequencing and cDNA library approach to the venomic analysis of Chinese spider Araneus ventricosus.

    Science.gov (United States)

    Duan, Zhigui; Cao, Rui; Jiang, Liping; Liang, Songping

    2013-01-14

    In past years, spider venoms have attracted increasing attention due to their extraordinary chemical and pharmacological diversity. The recently popularized proteomic method highly improved our ability to analyze the proteins in the venom. However, the lack of information about isolated venom proteins sequences dramatically limits the ability to confidently identify venom proteins. In the present paper, the venom from Araneus ventricosus was analyzed using two complementary approaches: 2-DE/Shotgun-LC-MS/MS coupled to MASCOT search and 2-DE/Shotgun-LC-MS/MS coupled to manual de novo sequencing followed by local venom protein database (LVPD) search. The LVPD was constructed with toxin-like protein sequences obtained from the analysis of cDNA library from A. ventricosus venom glands. Our results indicate that a total of 130 toxin-like protein sequences were unambiguously identified by manual de novo sequencing coupled to LVPD search, accounting for 86.67% of all toxin-like proteins in LVPD. Thus manual de novo sequencing coupled to LVPD search was proved an extremely effective approach for the analysis of venom proteins. In addition, the approach displays impeccable advantage in validating mutant positions of isoforms from the same toxin-like family. Intriguingly, methyl esterifcation of glutamic acid was discovered for the first time in animal venom proteins by manual de novo sequencing.

  13. Identification of Differentially Expressed Genes During Anther Abortion of Taigu Genic Male Sterile Wheat by Combining Suppression Subtractive Hybridization and cDNA Array

    Institute of Scientific and Technical Information of China (English)

    Qing-Shan Chang; Rong-Hua Zhou; Xiu-Ying Kong; Zeng-Liang Yu; Ji-Zeng Jia

    2006-01-01

    Taigu Genic Male Sterile Wheat (TGMSW; Triticum aestivum L.), a dominant genic male sterile germplasm, is of considerable value in the genetic improvement of wheat because of its stable inherence, complete male abortion, and high cross-fertilization rate. To identify specially transcribed genes in sterile anther, a suppression subtractive hybridization (SSH) library was constructed with sterile anther as the tester and fertile anther as the driver. A total of 2 304 SSH inserts amplified by polymerase chain reaction were arrayed using robotic printing. The cDNA arrays were hybridized with 32P-labeled probes prepared from the RNA of forward- and reverse-subtracted anthers. Ninety-six clones were scored as upregulated in sterile anthers compared with the corresponding fertile anthers and some clones were selected for sequencing and analysis in GenBank. Based on their putative functions, 87 non-redundant clones were classified into the following groups: (i) eight genes involved in metabolic processes; (ii) four material transportation genes;(iii) three signal transduction-associated genes; (iv) four stress response and senescence-associated protein genes; (v) seven other functional protein genes; (vi) five genes with no known function; and (vii)another 56 genes with no match to the databases. To test the hybridization efficiency, eight genes were selected and analyzed by Northern blot. The results of the present study provide a comprehensive overview of the genes and gene products involved in anther abortion in TGMSW.

  14. Transient B-Cell Depletion with Anti-CD20 in Combination with Proinsulin DNA Vaccine or Oral Insulin: Immunologic Effects and Efficacy in NOD Mice

    OpenAIRE

    Ghanashyam Sarikonda; Sowbarnika Sachithanantham; Yulia Manenkova; Tinalyn Kupfer; Amanda Posgai; Clive Wasserfall; Philip Bernstein; Laura Straub; Pagni, Philippe P.; Darius Schneider; Teresa Rodriguez Calvo; Marilyne Coulombe; Kevan Herold; Gill, Ronald G.; Mark Atkinson

    2013-01-01

    A recent type 1 diabetes (T1D) clinical trial of rituximab (a B cell-depleting anti-CD20 antibody) achieved some therapeutic benefit in preserving C-peptide for a period of approximately nine months in patients with recently diagnosed diabetes. Our previous data in the NOD mouse demonstrated that co-administration of antigen (insulin) with anti-CD3 antibody (a T cell-directed immunomodulator) offers better protection than either entity alone, indicating that novel combination therapies that i...

  15. A new method for species identification via protein-coding and non-coding DNA barcodes by combining machine learning with bioinformatic methods.

    Directory of Open Access Journals (Sweden)

    Ai-bing Zhang

    Full Text Available Species identification via DNA barcodes is contributing greatly to current bioinventory efforts. The initial, and widely accepted, proposal was to use the protein-coding cytochrome c oxidase subunit I (COI region as the standard barcode for animals, but recently non-coding internal transcribed spacer (ITS genes have been proposed as candidate barcodes for both animals and plants. However, achieving a robust alignment for non-coding regions can be problematic. Here we propose two new methods (DV-RBF and FJ-RBF to address this issue for species assignment by both coding and non-coding sequences that take advantage of the power of machine learning and bioinformatics. We demonstrate the value of the new methods with four empirical datasets, two representing typical protein-coding COI barcode datasets (neotropical bats and marine fish and two representing non-coding ITS barcodes (rust fungi and brown algae. Using two random sub-sampling approaches, we demonstrate that the new methods significantly outperformed existing Neighbor-joining (NJ and Maximum likelihood (ML methods for both coding and non-coding barcodes when there was complete species coverage in the reference dataset. The new methods also out-performed NJ and ML methods for non-coding sequences in circumstances of potentially incomplete species coverage, although then the NJ and ML methods performed slightly better than the new methods for protein-coding barcodes. A 100% success rate of species identification was achieved with the two new methods for 4,122 bat queries and 5,134 fish queries using COI barcodes, with 95% confidence intervals (CI of 99.75-100%. The new methods also obtained a 96.29% success rate (95%CI: 91.62-98.40% for 484 rust fungi queries and a 98.50% success rate (95%CI: 96.60-99.37% for 1094 brown algae queries, both using ITS barcodes.

  16. DNA damage in HBE cells induced by radon in combination with sodium arsenite%氡与亚砷酸钠致人永生化上皮细胞DNA损伤的联合毒作用

    Institute of Scientific and Technical Information of China (English)

    刘星; 聂继华; 陈志海; 金洹宇; 陈秋; 周新文; 焦旸; 童建; 安艳

    2013-01-01

    Objective To study DNA damage effect of radon in combination with sodium arsenite on HBE cells.Methods The HBE cells were exposed to various concentrations of radon (20 kBq/m3,0-75 min),sodium arsenite (0-50 mmol/L) and their combinations.Cell viability was tested by cell counting kit-8 (CCK-8).The combination index (CI) was calculated by different time exposed radon combined with different concentrations sodium arsenite.Single cell gel electrophoresis (SCGE) was applied for quantitative analysis of DNA damage.Results Radon with or without sodium arsenite inhibited the growth of HBE cells.Computed using median-effect principle,CI =0.41 indicated that radon and sodium arsenite had synergistic effect.Radon and sodium arsenite both could significantly increase tail DNA,tail moment and olive tail moment of HBE cells in SCGE compared with control(t =12.90-23.61,8.81-21.39,18.46-38.97,P<0.05; t=18.46-39.67,17.41-27.42,28.03-46.32,P<0.05).There was a dose-response relationship between radon level,sodium arsenite and DNA damage.The results of factorial analysis showed that there was a combination effect of DNA damage induced by radon and sodium arsenite (Ftail DNA% =76.07,P < 0.01 ; Ftail moment =290.51,P < 0.01 ; Foliver tail moment =273.46,P < 0.01).Conclusions There is a synergistic effect of DNA damage in HBE cells irradiated by radon in combination with sodium arsenite.%目的 探讨氡与亚砷酸钠联合染毒对人永生化上皮(HBE)细胞的DNA损伤作用.方法 以HBE细胞为研究对象,将HBE细胞暴露于20 000 Bq/m3氡浓度、不同时间(0~75 min)和不同浓度(0 ~ 50 000 μmol/L)亚砷酸钠以及联合作用,采用活细胞计数试剂盒(CCK-8)检测细胞存活率.通过中效原理,计算氡不同暴露时间和亚砷酸钠不同浓度联合作用联合指数(combination index,CI).以不同染氡时间(20 000 Bq/m3,10、20和40 min)和不同亚砷酸钠浓度(1.5、3.0和6.0 μmol/L)处理HBE细胞,应用单细胞凝胶电泳

  17. Proton-Induced X-ray Emission (PIXE Analysis and DNA-chain Break study in rat hepatocarcinogenesis: A possible chemopreventive role by combined supplementation of vanadium and beta-carotene

    Directory of Open Access Journals (Sweden)

    Kanjilal NB

    2005-05-01

    Full Text Available Abstract Combined effect of vanadium and beta-carotene on rat liver DNA-chain break and Proton induced X-ray emission (PIXE analysis was studied during a necrogenic dose (200 mg/kg of body weight of Diethyl Nitrosamine (DENA induced rat liver carcinogenesis. Morphological and histopathological changes were observed as an end point biomarker. Supplementation of vanadium (0.5 ppm ad libitum in drinking water and beta-carotene in the basal diet (120 mg/Kg of body weight were performed four weeks before DENA treatment and continued till the end of the experiment (16 weeks. PIXE analysis revealed the restoration of near normal value of zinc, copper, and iron, which were substantially altered when compared to carcinogen treated groups. Supplementation of both vanadium and beta-carotene four weeks before DENA injection was found to offer significant (64.73%, P

  18. Combining a nanosensor and ELISA for measurement of tyrosyl-dna phosphodiesterase 1 (tdp1) activity and protein amount in cell and tissue extract

    DEFF Research Database (Denmark)

    Jakobsen, Ann-Katrine; Stougaard, Magnus

    2015-01-01

    radiation and free radical-based genotoxins. TDP1 has been reported to be posttranslational regulated, and in nonsmall cell lung cancer (NSCLC) it has been reported that the enzyme activity of TDP1 can be upregulated without detectable upregulation in protein amount. Activity and protein analyses...... performed in the exact same aliquot of cell or tissue extract, the combined assay allows the use of minimal amount of cells or tissue and without additional incubation steps compared to the normal ELISA procedure. Measuring protein amount and activity within the same portion of extract also minimizes...... the risk of variation being introduced when comparing amount to activity. Since the assay only uses small amounts of extract, it requires no advanced equipment besides a plate reader, and can work in whole-cell or tissue extract. We expect that it could be useful for analysis of posttranslational...

  19. Comparison and combination of blood DNA methylation at smoking-associated genes and at lung cancer-related genes in prediction of lung cancer mortality.

    Science.gov (United States)

    Zhang, Yan; Breitling, Lutz P; Balavarca, Yesilda; Holleczek, Bernd; Schöttker, Ben; Brenner, Hermann

    2016-12-01

    Epigenome-wide association studies have established methylation patterns related to smoking, the major risk factor of lung cancer (LC), which are distinct from methylation profiles disclosed in LC patients. This study simultaneously investigated associations of smoking-associated and LC-related methylation markers with LC mortality. DNA methylation was determined by HM450K assay in baseline blood samples of 1,565 older adults in a population-based case-cohort study. The associations of 151 smoking-associated CpGs (smoCpGs) and 3,806 LC-related CpGs (caCpGs) with LC mortality were assessed by weighted Cox regression models, controlling for potential confounders. Multi-loci methylation scores were separately constructed based on smoCpGs and caCpGs. During a median follow-up of 13.8 years, 60 participants who had a first diagnosis of LC died from LC. The average time between sample collection and LC diagnosis was 5.8 years. Hypomethylation at 77 smoCpGs and 121 caCpGs, and hypermethylation at 4 smoCpGs and 66 caCpGs were associated with LC mortality. The associations were much stronger for smoCpGs than for caCpGs. Hazard ratios (95% CI) were 7.82 (2.91-21.00) and 2.27 (0.75-6.85), respectively, for participants in highest quartile of Score I (based on 81 smoCpGs) and Score II (based on 187 caCpGs), compared with participants in the corresponding lower three quartiles. Score I outperformed Score II, with an optimism-corrected C-index of 0.87 vs. 0.77. In conclusion, although methylation changes of both smoking-associated and LC-related genes are associated with LC mortality, only smoking-associated methylation markers predict LC mortality with high accuracy, and may thus serve as promising candidates to identify high risk populations for LC screening. PMID:27503000

  20. DNA immunization with fusion of CTLA-4 to hepatitis B virus (HBV core protein enhanced Th2 type responses and cleared HBV with an accelerated kinetic.

    Directory of Open Access Journals (Sweden)

    Ying Yin

    Full Text Available BACKGROUND: Typically, DNA immunization via the intramuscular route induces specific, Th1-dominant immune responses. However, plasmids expressing viral proteins fused to cytotoxic T lymphocyte antigen 4 (CTLA-4 primed Th2-biased responses and were able to induced effective protection against viral challenge in the woodchuck model. Thus, we addressed the question in the mouse model how the Th1/Th2 bias of primed immune responses by a DNA vaccine influences hepatitis B virus (HBV clearance. PRINCIPAL FINDINGS: Plasmids expressing HBV core protein (HBcAg or HBV e antigen and HBcAg fused to the extracellular domain of CTLA-4 (pCTLA-4-HBc, CD27, and full length CD40L were constructed. Immunizations of these DNA plasmids induced HBcAg-specific antibody and cytotoxic T-cell responses in mice, but with different characteristics regarding the titers and subtypes of specific antibodies and intensity of T-cell responses. The plasmid pHBc expressing HBcAg induced an IgG2a-dominant response while immunizations of pCTLA-4-HBc induced a balanced IgG1/IgG2a response. To assess the protective values of the immune responses of different characteristics, mice were pre-immunized with pCTLA-4-HBc and pHBc, and challenged by hydrodynamic injection (HI of pAAV/HBV1.2. HBV surface antigen (HBsAg and DNA in peripheral blood and HBcAg in liver tissue were cleared with significantly accelerated kinetics in both groups. The clearance of HBsAg was completed within 16 days in immunized mice while more than 50% of the control mice are still positive for HBsAg on day 22. Stronger HBcAg-specific T-cell responses were primed by pHBc correlating with a more rapid decline of HBcAg expression in liver tissue, while anti-HBs antibody response developed rapidly in the mice immunized with pCTLA-4-HBc, indicating that the Th1/Th2 bias of vaccine-primed immune responses influences the mode of viral clearance. CONCLUSION: Viral clearance could be efficiently achieved by Th1/Th2-balanced

  1. 血液净化联合DNA免疫吸附治疗重症系统性红斑狼疮的效果%Effect of blood purification combined with DNA immunoadsorption in treatment of severe systemic lupus erythematosus

    Institute of Scientific and Technical Information of China (English)

    鱼强; 刘岐焕; 程范军; 刘晓红; 张有顺

    2016-01-01

    Objective To analyze and explore the effect and safety of continuous blood purification combined with DNA immunoadsorp-tion,prednisone tablets and cyclophosphamide in the treatment of severe systemic lupus erythematosus. Methods 80 patients with severe systemic lupus erythematosus treated in our hospital were taken as the research objects and were averagely divided into the experimental group and the con-trol group. The control group were treated with DNA immunoadsorption,while the experimental group were treated with continuous blood purifica-tion combined with DNA immunoadsorption. The curative effect,renal function indexes and antinuclear antibody in the two groups were compara-tively analyzed. Results The total effective rate in the experimental group(95. 00℅ )was significantly higher than that in the control group(62. 50℅ )( P ﹤ 0. 05). 2 months of treatment later,the blood urea nitrogen(5. 31 ± 1. 25)mmol/ L and serum creatinine(98. 58 ± 21. 17)μmol/L of the experimental group were significantly better than those of the control group[(7. 23 ± 1. 33)mmol/ L and(128. 38 ± 25. 76)μmol/ L]( P ﹤ 0. 05). ANA(1. 62 ± 0. 41)and anti - ds - DHA antibody(0. 91 ± 0. 42)in the experimental group were significantly higher than those of the control group[(2. 21 ± 0. 69)and(1. 21 ± 0. 62)]( P ﹤ 0. 05). The incidence of adverse reactions in the experimental group(7. 50℅ ) was significantly lower than that in the control group(27. 50℅ )( P ﹤ 0. 05). Antinuclear antibody test group sensitivity of 30. 00℅ was signifi-cantly lower than the 62. 50℅ in the control group,the difference was statistically significant( P ﹤ 0. 05). Conclusion The effect of blood puri-fication combined with DNA immunoadsorption in treatment of severe systemic lupus erythematosus is significant. It can improve the renal function of patients,safe and reliable. It is worthy of promotion.%目的:探讨连续性血液净化联合 DNA 免疫吸附配合泼尼松片、环磷

  2. 宫颈液基细胞学与HPV-DNA在妇科普查中的作用%TCT and the combination of HPV-DNA in the census of women's diseases

    Institute of Scientific and Technical Information of China (English)

    罗业红; 沈琳菲; 邓廷坤; 刘莉

    2012-01-01

    Objective: to evaluate combine of the liquid-based cytology test and HPV-DNA test in checking gynecological diseases. Methods: This paper has selected 500 samples in all of the gynecological diseases tests form April 2009 to July 2012 randomly to Comparing the detection sensitivity and positive coincidence rate about liquid-based cytology test and HPV-DNA test. This paper adopts TBS grading criteria to comparing the results. The patients who cytological examination result were above ASCUS and HPV positive performed biopsy. Results: Cervical liquid-based cytology diagnostic results show:Normal range and benign reactive cell changing 407 cases (81.4%), HPV - DNA positive cytology diagnosis 106 cases (21.2%), and the pathological diagnosis positive cell 81 cases (16.2%). They are 54 cases confirmed for CIN-I、19 cases for CINII - III and 9 cases for SCC by biopsy. The result show it have huge difference between combine the liquid-based cytology test and HPV-DNA test and any method used alone. The samples are statistically significant (p<0.05).Conclusion: The application of TCT test combine with Higher-risk HPV test and TBS diagnostic criteria can improve the detection rate of cervical lesions.%目的评价液基细胞学检测和HPV-DNA检测的联合应用在妇女病普查中的作用.方法选取2009年4月-2012年07月在我院进行妇女病普查随机抽查的500例当成本文的研究对象,比较液基细胞学检测和HPV检测两种方法的检出敏感性及阳性符合率.诊断应用TBS分级标准,并对细胞学检查诊断为ASCUS以上者和HPV阳性者活检.结果宫颈液基细胞学诊断结果正常范围及良性反应性细胞改变407例(占81.4%),HPV-DNA阳性细胞学诊断106例(占21.2%),病理诊断结果阳性细胞81例(16.2%);经活检证实有54例为CIN-I,CINI-I I 19例,SCC 9例.单用TCT或HPV-DNA检测结果与联合检测比较差异显著,且具有统计学意义(P<0.05).结论 TCT检测结合高危型HPV检测技术的应

  3. Combining Push Pull Tracer Tests and Microbial DNA and mRNA Analysis to Assess In-Situ Groundwater Nitrate Transformations

    Science.gov (United States)

    Henson, W.; Graham, W. D.; Huang, L.; Ogram, A.

    2015-12-01

    Nitrogen transformation mechanisms in the Upper Floridan Aquifer (UFA) are still poorly understood because of karst aquifer complexity and spatiotemporal variability in nitrate and carbon loading. Transformation rates have not been directly measured in the aquifer. This study quantifies nitrate-nitrogen transformation potential in the UFA using single well push-pull tracer injection (PPT) experiments combined with microbial characterization of extracted water via qPCR and RT-qPCR of selected nitrate reduction genes. Tracer tests with chloride and nitrate ± carbon were executed in two wells representing anoxic and oxic geochemical end members in a spring groundwater contributing area. A significant increase in number of microbes with carbon addition suggests stimulated growth. Increases in the activities of denitrification genes (nirK and nirS) as measured by RT-qPCR were not observed. However, only microbes suspended in the tracer were obtained, ignoring effects of aquifer material biofilms. Increases in nrfA mRNA and ammonia concentrations were observed, supporting Dissimilatory Reduction of Nitrate to Ammonia (DNRA) as a reduction mechanism. In the oxic aquifer, zero order nitrate loss rates ranged from 32 to 89 nmol /L*hr with no added carbon and 90 to 240 nmol /L*hr with carbon. In the anoxic aquifer, rates ranged from 18 to 95 nmol /L*hr with no added carbon and 34 to 207 nmol /L*hr with carbon. These loss rates are low; 13 orders of magnitude less than the loads applied in the contributing area each year, however they do indicate that losses can occur in oxic and anoxic aquifers with and without carbon. These rates may include, ammonia adsorption, uptake, or denitrification in aquifer material biofilms. Rates with and without carbon addition for both aquifers were similar, suggesting aquifer redox state and carbon availability alone are insufficient to predict response to nutrient additions without characterization of microbial response. Surprisingly, these

  4. Investigation of Combined Detection of Fecal Occult Blood Test and Fecal DNA Test in the Opportunity Screening for Colorectal Cancer%联合检测粪隐血试验与粪便 DNA 在大肠癌机会性筛查中的探讨

    Institute of Scientific and Technical Information of China (English)

    钟选芳; 甘爱华; 张晓慧; 许岸高; 肖丹

    2015-01-01

    Objective To investigate the application and significance of opportunistic screening for colorectal cancer using combined detection of fecal occult blood test and fecal DNA test. Methods Forty-six patients with colorectal cancer and 30 normal volunteers were given with opportunistic screening,and were tested using immuno-chemical fecal occult blood tests(IFOBT),as well as the APC,P53 and K-ras mutations in the feces were detected by using polymerase chain reaction single strand conformation polymorphism(PCR-SSCP). Results The positive rate of IFOBT was 45. 7%(21 / 46)and 13. 3%(4 / 30)in the colorectal cancer group and the normal group,respec-tively. Meanwhile,the mutation rate of APC,P53,K-ras genes in the colorectal cancer group were 58. 7%(27 / 46), 65. 2%(30 / 46)and 60. 9%(28 / 46);and were 3. 3%(1 / 30),0. 0%(0 / 30)and 0. 0%(0 / 30)in the normal group. There was no significant difference in the sensitivity and specificity between IFOBT and fecal DNA screening of colorectal cancer,and the sensitivity of joint detection was higher than any single detection method. Conclusion The combined detection of fecal occult blood test and fecal DNA can raise the efficacy of the colorectal cancer screening,it can be used to become an effective noninvasive method of colorectal cancer in the opportunistic screen-ing of early diagnosis and screening.%目的:探讨联合检测粪隐血试验与粪便 DNA 用于大肠癌机会性筛查的应用和意义。方法入组行机会性筛查的46例大肠癌患者和30例正常者的粪便,分别应用免疫法粪便隐血试验(IFOBT)2次,应用PCR-SSCP 方法检测粪便中 APC、P53和 K-ras 基因突变情况。结果大肠癌组和正常组粪便 IFOBT 的阳性率分别为45.7%(21/46)和13.3%(4/30),APC、P53、K-ras 的突变率分别为58.7%(27/46)、65.2%(30/46)、60.9%(28/46)和3.3%(1/30)、0.0%(0/30)、0.0%(0/30),IFOBT 和粪便 DNA 筛查大肠癌的敏感性和特异性差

  5. Isolation, cDNA sequence analysis and tissue expression profile of a novel swine gene differentially expressed in the Longissimus dorsi muscle tissues from Large White × Meishan cross combination

    Institute of Scientific and Technical Information of China (English)

    LIU Yonggang; LEI Minggang; XIONG Yuanzhu; DENG Changyan

    2005-01-01

    In order to study the molecular mechanism of heterosis in pigs, the mRNA differential display technique was performed to investigate the differences in gene expression in the Longissimus dorsi muscle tissues from Large White × Meishan cross combination.One novel gene differentially expressed between the hybrids and the purebreds was isolated and subsequently identified using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and its complete cDNA sequence was obtained using the rapid amplification of cDNA ends (RACE) method. The nucleotide sequence of the gene is not homologous to any of the known porcine genes. The sequence prediction revealed that the open reading frame of this gene encodes a protein of 188 amino acids that contains the putative conserved domain of the PRA1 family protein and this protein has high homology with the PRA1 family protein 3 of three species-rat (88 % ), human(88 % ), and mouse (87 % ), -so that it can be defined as swine PRA1 family protein 3. The phylogenetic tree analysis revealed that the swine PRA1 family protein 3 has a closer genetic relationship with the human PRA1 family protein 3 than with those of mouse and rat.The tissue expression analysis indicated that swine PRA1family protein 3 gene is highly-expressed in muscle and fat, moderately in spleen,weakly in heart, kidney, ovary, lung, and almost not expressed in small intestine and liver. The function of this gene and the relationship between this gene and heterosis are also discussed.

  6. Dna Sequencing

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  7. DNA-Based Kinship Analysis

    OpenAIRE

    Maguire, Christopher; Woodward, Michael

    2008-01-01

    Relatedness between individuals and groups can be investigated using DNA markers. A child’s DNA profile is a combination of alleles passed down from the father and mother. This means that relationships can be investigated between alleged family members. DNA profiling is commonly used to test for potential paternity, parentage and sibship (whether people are related as brothers or sisters) relationships. In many forensic cases more complex relationships have to be considered.

  8. Premeltons in DNA.

    Science.gov (United States)

    Sobell, Henry M

    2016-03-01

    Premeltons are examples of emergent-structures (i.e., structural-solitons) that arise spontaneously in DNA due to the presence of nonlinear-excitations in its structure. They are of two kinds: B-B (or A-A) premeltons form at specific DNA-regions to nucleate site-specific DNA melting. These are stationary and, being globally-nontopological, undergo breather-motions that allow drugs and dyes to intercalate into DNA. B-A (or A-B) premeltons, on the other hand, are mobile, and being globally-topological, act as phase-boundaries transforming B- into A-DNA during the structural phase-transition. They are not expected to undergo breather motions. A key feature of both types of premeltons is the presence of an intermediate structural-form in their central regions (proposed as being a transition-state intermediate in DNA-melting and in the B- to A-transition), which differs from either A- or B-DNA. Called beta-DNA, this is both metastable and hyperflexible--and contains an alternating sugar-puckering pattern along the polymer backbone combined with the partial unstacking (in its lower energy-forms) of every-other base-pair. Beta-DNA is connected to either B- or to A-DNA on either side by boundaries possessing a gradation of nonlinear structural-change, these being called the kink and the antikink regions. The presence of premeltons in DNA leads to a unifying theory to understand much of DNA physical chemistry and molecular biology. In particular, premeltons are predicted to define the 5' and 3' ends of genes in naked-DNA and DNA in active-chromatin, this having important implications for understanding physical aspects of the initiation, elongation and termination of RNA-synthesis during transcription. For these and other reasons, the model will be of broader interest to the general-audience working in these areas. The model explains a wide variety of data, and carries with it a number of experimental predictions--all readily testable--as will be described in this review.

  9. Effects of oxaliplatin on DNA condensation

    Institute of Scientific and Technical Information of China (English)

    JU HaiPeng; ZHANG HongYan; LI Wei; WANG PengYe

    2014-01-01

    In this paper the interactions between DNA and anti-cancer drug oxaliplatin were investigated by using magnetic tweezers.The dynamics of DNA condensation due to oxaliplatin was traced under various forces.It is found that torsion constraint in DNA enhances the ability of oxaliplatin for shortening DNA.The transplatin helps oxaliplatin combine to DNA and increase the rate of DNA condensation.All these results are consistent to the previously proposed model and are helpful for further investigation of interaction between DNA and oxaliplatin.

  10. Random amplified polymorphic DNA combined with microfluidic chips in the identification of Malassezia species%随机扩增多态性DNA结合微流芯片鉴定马拉色菌的初步研究

    Institute of Scientific and Technical Information of China (English)

    王韵茹; 章强强

    2012-01-01

    stable and clear amplification bands than that with S24.Positive bands of different sizes were repetitively obtained by using microfluidic chips,with interspecies and intraspecies polymorphisms observed in all the strains.On the basis of DNA sequencing,microfluidic chips and RAPD-PCR could be used to successfully distinguish the following eight species,i.e.,M.furfur,M.sympodialis,M.globosa,M.pacbydermatis,M.slooffiae,M.Japonica,M.yamatoensis and M.dermatis.Conclusions As a rapid,high-throughput and high-sensitivity method,microfluidic chips combined with RAPD-PCR shows an advantage for analyzing interspecies genetic diversity,genetic relationship of Malassezia species,as well as for identifying new Malassezia species.

  11. Individual and Combined Expression of DNA Damage Response Molecules PARP1, γH2AX, BRCA1, and BRCA2 Predict Shorter Survival of Soft Tissue Sarcoma Patients

    Science.gov (United States)

    Park, See-Hyoung; Park, Hye Jeong; Wang, Sung Il; Park, Ho Sung; Lee, Ho; Kwon, Keun Sang; Moon, Woo Sung; Lee, Dong Geun; Kim, Jung Ryul; Jang, Kyu Yun

    2016-01-01

    DNA damage response (DDR) molecules are protective against genotoxic stresses. DDR molecules are also involved in the survival of cancer cells in patients undergoing anti-cancer therapies. Therefore, DDR molecules are potential markers of cancer progression in addition to being potential therapeutic targets. In this study, we evaluated the immunohistochemical expression of PARP1, γH2AX, BRCA1, and BRCA2 and their prognostic significance in 112 cases of soft tissue sarcoma (STS). The expression of PARP1, γH2AX, BRCA1, and BRCA2 were significantly associated with each other and were associated with higher tumor stage and presence of distant metastasis. The expression of PARP1, γH2AX, and BRCA2 were significantly associated with shorter disease-specific survival (DSS) and event-free survival (EFS) by univariate analysis. BRCA1 expression was associated with shorter DSS. Multivariate analysis revealed the expression of PARP1 and γH2AX to be independent indicators of poor prognosis of DSS and EFS. BRCA2 expression was an independent indicator of poor prognosis of DSS. In addition, the combined expressional patterns of PARP1, γH2AX, BRCA1, and BRCA2 (CSddrm) were independent prognostic predictors of DSS (P DSS rate of the CSddrm-low, CSddrm-intermediate, and CSddrm-high subgroups were 81%, 26%, and 0%, respectively. In conclusion, this study demonstrates that the individual and combined expression patterns of the DDR molecules PARP1, γH2AX, BRCA1, and BRCA2 could be predictive of the prognosis of STS patients and suggests that controlling the activity of these DDR molecules could be employed in new therapeutic stratagems for the treatment of STS. PMID:27643881

  12. Detection of DNA virus in respiratory samples of children by multiplex PCR combined with RDB%PCR-RDB技术快速检测儿童急性呼吸道感染DNA病毒的研究

    Institute of Scientific and Technical Information of China (English)

    田海清; 曾跃红; 王新华; 周勇军

    2015-01-01

    Objective To develop multiplex polymerase chain reaction (PCR) combined with reversedotblothy bridization (RDB) method for detection of DNA virus in respiratory samples,and provide a surveillance and rapid diagnosis tool of acute viral respiratory infection.Methods We designed multiple PCR primers and the probes referenced to virus nucleic acid sequences in the National Center for Biotechnology Information (NCBI) database,and fixed specific oligonucleotide probes on the nylon membrane.After multiple PCR amplification of virus DNA of human bocavirus (hBOV),karolinska Institutet (KI),adenovirus (AdV),Washington University polyomaviros (WUPyV),and human parvovirus B19 (HPVBI9),the denaturalized amplification products were hybridized with various specific probes,followed by visualization and analysis of the results.The sensitivity and specificity were tested.At the same time,108 cases of clinical specimens of multiple PCR products were analyzed by reverse spot hybridization detection,and compared to the results of culture method.Results The specific probes of multiple PCR-RDB only hybridized with corresponding amplification products without cross-hybridization reaction with other pathogen.The sensitivity of RDB hybridization was 1 colony-forming units (CFU).The positive rate of 34.26% (37 cases out of 108 cases) with PCR-RDB method was significandy higher than that 27.78% (30 cases out of 108 cases) with common test method.Conclusions The multiplex PCR combined with RDB might become a rapid and simple method to detect the DNA virus in respiratory samples,which might be a promising tool for clinical application.%目的 建立多重PCR反向斑点杂交方法(PCR-RDB)技术快速检测儿童急性呼吸道感染DNA病毒,以便及时监测并快速诊断急性病毒性呼吸道感染.方法 参照国家生物技术信息中心(NCBI)数据库病毒核酸序列设计多重PCR引物和探针,将特异的寡核苷酸探针固定于尼龙膜上.利用多重PCR对人博

  13. Individual and Combined Expression of DNA Damage Response Molecules PARP1, γH2AX, BRCA1, and BRCA2 Predict Shorter Survival of Soft Tissue Sarcoma Patients

    Science.gov (United States)

    Park, See-Hyoung; Park, Hye Jeong; Wang, Sung Il; Park, Ho Sung; Lee, Ho; Kwon, Keun Sang; Moon, Woo Sung; Lee, Dong Geun; Kim, Jung Ryul; Jang, Kyu Yun

    2016-01-01

    DNA damage response (DDR) molecules are protective against genotoxic stresses. DDR molecules are also involved in the survival of cancer cells in patients undergoing anti-cancer therapies. Therefore, DDR molecules are potential markers of cancer progression in addition to being potential therapeutic targets. In this study, we evaluated the immunohistochemical expression of PARP1, γH2AX, BRCA1, and BRCA2 and their prognostic significance in 112 cases of soft tissue sarcoma (STS). The expression of PARP1, γH2AX, BRCA1, and BRCA2 were significantly associated with each other and were associated with higher tumor stage and presence of distant metastasis. The expression of PARP1, γH2AX, and BRCA2 were significantly associated with shorter disease-specific survival (DSS) and event-free survival (EFS) by univariate analysis. BRCA1 expression was associated with shorter DSS. Multivariate analysis revealed the expression of PARP1 and γH2AX to be independent indicators of poor prognosis of DSS and EFS. BRCA2 expression was an independent indicator of poor prognosis of DSS. In addition, the combined expressional patterns of PARP1, γH2AX, BRCA1, and BRCA2 (CSddrm) were independent prognostic predictors of DSS (P stratagems for the treatment of STS. PMID:27643881

  14. DNA-PK assay

    Science.gov (United States)

    Anderson, Carl W.; Connelly, Margery A.

    2004-10-12

    The present invention provides a method for detecting DNA-activated protein kinase (DNA-PK) activity in a biological sample. The method includes contacting a biological sample with a detectably-labeled phosphate donor and a synthetic peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamine-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and, (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. A compostion and a kit for the detection of DNA-PK activity are also provided. Methods for detecting DNA, protein phosphatases and substances that alter the activity of DNA-PK are also provided. The present invention also provides a method of monitoring protein kinase and DNA-PK activity in living cells. -A composition and a kit for monitoring protein kinase activity in vitro and a composition and a kit for monitoring DNA-PK activities in living cells are also provided. A method for identifying agents that alter protein kinase activity in vitro and a method for identifying agents that alter DNA-PK activity in living cells are also provided.

  15. Potential predictive role of chemotherapy-induced changes of soluble CD40 ligand in untreated advanced pancreatic ductal adenocarcinoma.

    Science.gov (United States)

    Azzariti, Amalia; Brunetti, Oronzo; Porcelli, Letizia; Graziano, Giusi; Iacobazzi, Rosa Maria; Signorile, Michele; Scarpa, Aldo; Lorusso, Vito; Silvestris, Nicola

    2016-01-01

    Pancreas ductal adenocarcinoma lacks predictive biomarkers. CD40 is a member of the tumor necrosis factor superfamily. CD40-sCD40L interaction is considered to contribute to the promotion of tumor cell growth and angiogenesis. The aim of the present study was to investigate the role of serum sCD40L as a predictor in metastatic pancreatic cancer. We evaluated 27 consecutive pancreatic cancer patients treated with FOLFIRINOX (21 patients) or gemcitabine plus nab-paclitaxel combination (six patients). The sCD40L level was measured in serum by enzyme-linked immunosorbent assay at baseline, at first evaluation (all patients), and at time to progression (18 patients). The radiological response was evaluated according to the Response Evaluation Criteria in Solid Tumors, Version 1.1. The Wilcoxon signed-rank test was used to compare pre-post treatment sCD40L levels with respect to clinical response, while Pearson's correlation coefficient was used for the correlation between sCD40L and CA19.9 pre- and post-treatment. The Kruskal-Wallis test was also conducted for further comparisons. We observed a statistically significant reduction in the sCD40L level after 3 months of treatment in patients with partial response (11,718.05±7,097.13 pg/mL vs 4,689.42±5,409.96 pg/mL; Pvariation percentage (Pearson's correlation coefficient =0.52; P<0.05). Our data suggest a possible predictive role of sCD40L in pancreatic cancer patients, similar to CA19.9. PMID:27555786

  16. Immunological competence of a DNA vaccine containing human papillomavirus type 16 E2 combined with IL-12%HPV16 E2与IL-12联合基因疫苗的免疫效果初步研究

    Institute of Scientific and Technical Information of China (English)

    唐双阳; 李乐; 余敏君; 粟盛梅; 蔡恒玲; 周良; 万艳平

    2012-01-01

    vaccine group), and the pcDNA3. 1( + )/HPV16 E2 + pcDNA3. 1( + )/IL-12 Group (the combined DNA vaccine group). The mice were injected every four times every two weeks. The sera of mice were collected and stored at — 20 ℃ the day before every immunization or the day before sacrifice. Spleen cells were cultured from the spleens of the mice. ELISA was used to measure the serum-specific IgG antibody and IFN-γ or IL-4 levels in the supernatant from cultured spleen cells. The proliferative response of the spleen cells was measured with an MTT assay. Results The A45O value for IgG in the final immunized sera from the combined DNA vaccine group and the single DNA vaccine group was significantly higher than that from the pcDNA3. 1( + ) Group and PBS Group (P0. 05) in the A450 value for the combined DNA vaccine group and the single DNA vaccine group at any of the vaccination times. IFN-γ and IL-4 levels in the supernatant of he spleen lymphocytes from the pcDNA3. 1( + )/IL-12 Group, the combined DNA vaccine group, and the single DNA vaccine group were significantly higher than those from the pcDNA3. 1( + ) Group and PBS Group (P0. 05). The stimulation index (SI) of the spleen cells from the pcDNA3. 1( + )/IL-12 Group, the combined DNA vaccine group, and the single DNA vaccine group was higher than that from the pcDNA3. 1( + ) Group and PBS Group (P0. 05). Conclusion The HPV16 E2 vaccine may stimulate the level of specific antibodies and increase the level of IFN-γ. A combination DNA vaccine may have a more potent immune effect.

  17. Radiation response and regulation of apoptosis induced by a combination of TRAIL and CHX in cells lacking mitochondrial DNA: A role for NF-{kappa}B-STAT3-directed gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, Vladimir N., E-mail: vni3@columbia.edu; Ghandhi, Shanaz A.; Zhou, Hongning; Huang, Sarah X.; Chai, Yunfei; Amundson, Sally A.; Hei, Tom K.

    2011-07-01

    Mitochondrial DNA depleted ({rho}{sup 0}) human skin fibroblasts (HSF) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NF-{kappa}B and STAT3 signaling pathways, and by decreased activity of mitochondrial death pathway, compared to the parental {rho}{sup +} HSF. In contrast, the extrinsic TRAIL/TRAIL-Receptor mediated death pathway remained highly active, and exogenous TRAIL in a combination with cycloheximide (CHX) induced higher levels of apoptosis in {rho}{sup 0} cells compared to {rho}{sup +} HSF. Global gene expression analysis using microarray and qRT-PCR demonstrated that mRNA expression levels of many growth factors and their adaptor proteins (FGF13, HGF, IGFBP4, IGFBP6, and IGFL2), cytokines (IL6, {Oota}L17{Beta}, {Oota}L18, {Oota}L19, and {Oota}L28{Beta}) and cytokine receptors (IL1R1, IL21R, and IL31RA) were substantially decreased after mitochondrial DNA depletion. Some of these genes were targets of NF-{kappa}B and STAT3, and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation further induced expression of several NF-{kappa}B/STAT3 target genes, including IL1A, IL1B, IL6, PTGS2/COX2 and MMP12, in {rho}{sup +} HSF, but this response was substantially decreased in {rho}{sup 0} HSF. Suppression of the IKK-NF-{kappa}B pathway by the small molecular inhibitor BMS-345541 and of the JAK2-STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated {rho}{sup +} HSF. Inhibitory antibodies against IL6, the main activator of JAK2-STAT3 pathway, added into the cell media, also increased TRAIL-induced apoptosis in HSF, especially after alpha-irradiation. Collectively, our results indicated that NF-{kappa}B activation was partially lost in {rho}{sup 0} HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-{kappa}B targets, further suppressing IL6

  18. Microarray Detection of Duplex and Triplex DNA Binders with DNA-Modified Gold Nanoparticles

    OpenAIRE

    Lytton-Jean, Abigail K. R.; Han, Min Su; Mirkin, Chad A.

    2007-01-01

    We have designed a chip-based assay, using microarray technology, for determining the relative binding affinities of duplex and triplex DNA binders. This assay combines the high discrimination capabilities afforded by DNA-modified Au nanoparticles with the high-throughput capabilities of DNA microarrays. The detection and screening of duplex DNA binders are important because these molecules, in many cases, are potential anticancer agents as well as toxins. Triplex DNA binders are also promisi...

  19. DNA methylation

    DEFF Research Database (Denmark)

    Williams, Kristine; Christensen, Jesper; Helin, Kristian

    2012-01-01

    DNA methylation is involved in key cellular processes, including X-chromosome inactivation, imprinting and transcriptional silencing of specific genes and repetitive elements. DNA methylation patterns are frequently perturbed in human diseases such as imprinting disorders and cancer. The recent d...

  20. DNA glue

    DEFF Research Database (Denmark)

    Filichev, Vyacheslav V; Astakhova, Irina V.; Malakhov, Andrei D.;

    2008-01-01

    Significant alterations in thermal stability of parallel DNA triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in the structure of phenylmethylglycerol inserted as a bulge into DNA (TINA). Insertions of two ortho-TINAs as a pseudo...

  1. Enhancing nanopore sensing with DNA nanotechnology

    Science.gov (United States)

    Keyser, Ulrich F.

    2016-02-01

    Nanopores are on the brink of fundamentally changing DNA sequencing. At the same time, DNA origami provides unprecedented freedom in molecular design. Here, I suggest why a combination of solid-state nanopores and DNA nanotechnology will lead to exciting new experiments.

  2. PDA: Pooled DNA analyzer

    Directory of Open Access Journals (Sweden)

    Lin Chin-Yu

    2006-04-01

    Full Text Available Abstract Background Association mapping using abundant single nucleotide polymorphisms is a powerful tool for identifying disease susceptibility genes for complex traits and exploring possible genetic diversity. Genotyping large numbers of SNPs individually is performed routinely but is cost prohibitive for large-scale genetic studies. DNA pooling is a reliable and cost-saving alternative genotyping method. However, no software has been developed for complete pooled-DNA analyses, including data standardization, allele frequency estimation, and single/multipoint DNA pooling association tests. This motivated the development of the software, 'PDA' (Pooled DNA Analyzer, to analyze pooled DNA data. Results We develop the software, PDA, for the analysis of pooled-DNA data. PDA is originally implemented with the MATLAB® language, but it can also be executed on a Windows system without installing the MATLAB®. PDA provides estimates of the coefficient of preferential amplification and allele frequency. PDA considers an extended single-point association test, which can compare allele frequencies between two DNA pools constructed under different experimental conditions. Moreover, PDA also provides novel chromosome-wide multipoint association tests based on p-value combinations and a sliding-window concept. This new multipoint testing procedure overcomes a computational bottleneck of conventional haplotype-oriented multipoint methods in DNA pooling analyses and can handle data sets having a large pool size and/or large numbers of polymorphic markers. All of the PDA functions are illustrated in the four bona fide examples. Conclusion PDA is simple to operate and does not require that users have a strong statistical background. The software is available at http://www.ibms.sinica.edu.tw/%7Ecsjfann/first%20flow/pda.htm.

  3. Cryptography with DNA binary strands.

    Science.gov (United States)

    Leier, A; Richter, C; Banzhaf, W; Rauhe, H

    2000-06-01

    Biotechnological methods can be used for cryptography. Here two different cryptographic approaches based on DNA binary strands are shown. The first approach shows how DNA binary strands can be used for steganography, a technique of encryption by information hiding, to provide rapid encryption and decryption. It is shown that DNA steganography based on DNA binary strands is secure under the assumption that an interceptor has the same technological capabilities as sender and receiver of encrypted messages. The second approach shown here is based on steganography and a method of graphical subtraction of binary gel-images. It can be used to constitute a molecular checksum and can be combined with the first approach to support encryption. DNA cryptography might become of practical relevance in the context of labelling organic and inorganic materials with DNA 'barcodes'.

  4. The DNA injury-repair of esophageal cancer EC109 cells to diallyl trisulfide (DATS) combining radiation%二烯丙基三硫化物联合辐射对食管癌EC109细胞DNA损伤及修复影响

    Institute of Scientific and Technical Information of China (English)

    Yang Zhang; Hongbing Ma

    2012-01-01

    Objective: The aim of our study was to investigate the effect of diallyl trisulfide (DATS) combining radiation on DNA injury-repair of Esophageal cancer EC109 cells. Methods: Using 10 and 20 μg/mL DATS on EC109 cells, and taking X-ray radiation 24 h later. Investigate the radiosensitization effect of DATS on EC109 cells by clone formation, and the mechanism of DNA injury-repair by Comet Assay. Results: The clone formation resulted that DATS had radiosensitization effect on EC109 cells. Radiosensitization enhancement ratios of 10 and 20 μg/mL DATS in combination with radiation were 1.55,1.64 (Do) and 1.43,1.75 (Dq) respectively. In the comet assay, the TM (tail moments) of 20 μg/mL DATS combining radiation group lines at 0 h, 2 h, 6 h and 24 h were 7.16 ± 2.61,3.65 ± 2.06,2.09 ± 0.83,1.45 ± 1.37 respectively. They were slightly increased than radiation group (0.95 ± 0.65, 0.11 ± 0.07, 0.1 ± 0.05, 0.11 ± 0.08) and DATS group (1.81 ± 1.23,1.58 ± 1.40, 0.45 ± 0.25,0.60 ± 0.40) (P < 0.01). The result showed that DATS combining radiation had the effect of increasing DNA damage and inhibiting DNA repair on EC109 cells. Conclusion: DATS has radiosensitization effect on Esophageal cancer EC109 cells. And the effect is probably related with DNA injury-repair.

  5. [DNA computing].

    Science.gov (United States)

    Błasiak, Janusz; Krasiński, Tadeusz; Popławski, Tomasz; Sakowski, Sebastian

    2011-01-01

    Biocomputers can be an alternative for traditional "silicon-based" computers, which continuous development may be limited due to further miniaturization (imposed by the Heisenberg Uncertainty Principle) and increasing the amount of information between the central processing unit and the main memory (von Neuman bottleneck). The idea of DNA computing came true for the first time in 1994, when Adleman solved the Hamiltonian Path Problem using short DNA oligomers and DNA ligase. In the early 2000s a series of biocomputer models was presented with a seminal work of Shapiro and his colleguas who presented molecular 2 state finite automaton, in which the restriction enzyme, FokI, constituted hardware and short DNA oligomers were software as well as input/output signals. DNA molecules provided also energy for this machine. DNA computing can be exploited in many applications, from study on the gene expression pattern to diagnosis and therapy of cancer. The idea of DNA computing is still in progress in research both in vitro and in vivo and at least promising results of these research allow to have a hope for a breakthrough in the computer science. PMID:21735816

  6. DNA probes

    International Nuclear Information System (INIS)

    The creation of DNA probes for detection of specific nucleotide segments differs from ligand detection in that it is a chemical rather than an immunological reaction. Complementary DNA or RNA is used in place of the antibody and is labelled with 32P. So far, DNA probes have been successfully employed in the diagnosis of inherited disorders, infectious diseases, and for identification of human oncogenes. The latest approach to the diagnosis of communicable and parasitic infections is based on the use of deoxyribonucleic acid (DNA) probes. The genetic information of all cells is encoded by DNA and DNA probe approach to identification of pathogens is unique because the focus of the method is the nucleic acid content of the organism rather than the products that the nucleic acid encodes. Since every properly classified species has some unique nucleotide sequences that distinguish it from every other species, each organism's genetic composition is in essence a finger print that can be used for its identification. In addition to this specificity, DNA probes offer other advantages in that pathogens may be identified directly in clinical specimens

  7. Cellular factors required for papillomavirus DNA replication.

    OpenAIRE

    Melendy, T; Sedman, J; Stenlund, A

    1995-01-01

    In vitro replication of papillomavirus DNA has been carried out with a combination of purified proteins and partially purified extracts made from human cells. DNA synthesis requires the viral E1 protein and the papillomavirus origin of replication. The E2 protein stimulates DNA synthesis in a binding site-independent manner. Papillomavirus DNA replication is also dependent on the cellular factors replication protein A, replication factor C, and proliferating-cell nuclear antigen as well as a ...

  8. Human DNA Ligase III Recognizes DNA Ends by Dynamic Switching between Two DNA-Bound States

    Energy Technology Data Exchange (ETDEWEB)

    Cotner-Gohara, Elizabeth; Kim, In-Kwon; Hammel, Michal; Tainer, John A.; Tomkinson, Alan E.; Ellenberger, Tom (Scripps); (Maryland-MED); (WU-MED); (LBNL)

    2010-09-13

    Human DNA ligase III has essential functions in nuclear and mitochondrial DNA replication and repair and contains a PARP-like zinc finger (ZnF) that increases the extent of DNA nick joining and intermolecular DNA ligation, yet the bases for ligase III specificity and structural variation among human ligases are not understood. Here combined crystal structure and small-angle X-ray scattering results reveal dynamic switching between two nick-binding components of ligase III: the ZnF-DNA binding domain (DBD) forms a crescent-shaped surface used for DNA end recognition which switches to a ring formed by the nucleotidyl transferase (NTase) and OB-fold (OBD) domains for catalysis. Structural and mutational analyses indicate that high flexibility and distinct DNA binding domain features in ligase III assist both nick sensing and the transition from nick sensing by the ZnF to nick joining by the catalytic core. The collective results support a 'jackknife model' in which the ZnF loads ligase III onto nicked DNA and conformational changes deliver DNA into the active site. This work has implications for the biological specificity of DNA ligases and functions of PARP-like zinc fingers.

  9. Human DNA ligase III recognizes DNA ends by dynamic switching between two DNA-bound states.

    Science.gov (United States)

    Cotner-Gohara, Elizabeth; Kim, In-Kwon; Hammel, Michal; Tainer, John A; Tomkinson, Alan E; Ellenberger, Tom

    2010-07-27

    Human DNA ligase III has essential functions in nuclear and mitochondrial DNA replication and repair and contains a PARP-like zinc finger (ZnF) that increases the extent of DNA nick joining and intermolecular DNA ligation, yet the bases for ligase III specificity and structural variation among human ligases are not understood. Here combined crystal structure and small-angle X-ray scattering results reveal dynamic switching between two nick-binding components of ligase III: the ZnF-DNA binding domain (DBD) forms a crescent-shaped surface used for DNA end recognition which switches to a ring formed by the nucleotidyl transferase (NTase) and OB-fold (OBD) domains for catalysis. Structural and mutational analyses indicate that high flexibility and distinct DNA binding domain features in ligase III assist both nick sensing and the transition from nick sensing by the ZnF to nick joining by the catalytic core. The collective results support a "jackknife model" in which the ZnF loads ligase III onto nicked DNA and conformational changes deliver DNA into the active site. This work has implications for the biological specificity of DNA ligases and functions of PARP-like zinc fingers. PMID:20518483

  10. DNA data

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Raw DNA chromatogram data produced by the ABI 373, 377, 3130 and 3730 automated sequencing machines in ABI format. These are from fish (primarily Sebastes spp.,...

  11. Phylogeny of Deltocephalinae (Hemiptera: Cicadellidae)from China based on partial 16S rDNA and 28S rDNA D2 sequences combined with morphological characters%基于16S rDNA和28S rDNA D2基因序列与形态特征联合分析的中国角顶叶蝉亚科系统发育研究(半翅目:叶蝉科)

    Institute of Scientific and Technical Information of China (English)

    戴仁怀; 陈学新; 李子忠

    2008-01-01

    The phylogeny of 19 genera of Deltocephalinae leafhoppers was analyzed based on 50 adult morphological characters combined with nucleotide sequences of the mitochondrial 16S rDNA and nuclear 28S D2 rDNA genes. One species of Typhlocybinae was included as outgroup. Parsimonian, distance and Bayesian methods were used to estimate the phylogenetic relationships. The topology of the phylogenetic trees generated with different methods was quite similar. We partially resolved the morphologically-defined tribes and the relationships among 19 genera of Deltocephalinae. The genus Macrosteles was well supported to occupy a basal position in the study, so the most primary tribe in Deltocephalinae might be Macrostelini. The phylogenetic analysis trees put all genera of Deltocephalini but Nakaharanus onto a single lineage. The genus Balclutha, corresponding to the tribe Balclnthini,remains unsolved in our analyses. The Euscelini might be a polyphyletic group in the analysis. Analytical result recovered Athysanini and Paralimnini as monophyletic clades. The clade Phlogotettix and Scaphoideus-Nakaharanus was constantly resolved using different methods. We suggested that Scaphoideus, Nakaharanus and Phlogotettix should be included in or into Scaphoideini. But the results resolved poorly the taxonomic status of Xestoeephalini overall.%首次在国内利用28s rDNA D2区段和16s rDNA基因序列,结合50个形态特征对角顶叶蝉亚科(Deltocephalinae)[半翅目(Hemiptera):叶蝉科(cicadellidae)]19个属进行系统发育分析研究.从无水乙醇浸泡保存的标本中提取基因组DNA并扩增了19个内群和1种外群Tyhlocybinae[半翅目(Hemiptera):叶蝉科(cicadelIidae)]种类的28s rDNA D2基因片段并测序,同时扩增了16s rDNA基因片段并测序11条,采用了GenBank中1个种类的16S rDNA同源序列.采用PAuP*4.O和MrBayes3.0两个分析软件和3种建树方法,利用同源28s D2 rDNA和16srDNA两个基因序列与形态特征结合进行系统发

  12. DNA expressions - A formal notation for DNA

    NARCIS (Netherlands)

    Vliet, Rudy van

    2015-01-01

    We describe a formal notation for DNA molecules that may contain nicks and gaps. The resulting DNA expressions denote formal DNA molecules. Different DNA expressions may denote the same molecule. Such DNA expressions are called equivalent. We examine which DNA expressions are minimal, which

  13. Programmable Quantitative DNA Nanothermometers.

    Science.gov (United States)

    Gareau, David; Desrosiers, Arnaud; Vallée-Bélisle, Alexis

    2016-07-13

    Developing molecules, switches, probes or nanomaterials that are able to respond to specific temperature changes should prove of utility for several applications in nanotechnology. Here, we describe bioinspired strategies to design DNA thermoswitches with programmable linear response ranges that can provide either a precise ultrasensitive response over a desired, small temperature interval (±0.05 °C) or an extended linear response over a wide temperature range (e.g., from 25 to 90 °C). Using structural modifications or inexpensive DNA stabilizers, we show that we can tune the transition midpoints of DNA thermometers from 30 to 85 °C. Using multimeric switch architectures, we are able to create ultrasensitive thermometers that display large quantitative fluorescence gains within small temperature variation (e.g., > 700% over 10 °C). Using a combination of thermoswitches of different stabilities or a mix of stabilizers of various strengths, we can create extended thermometers that respond linearly up to 50 °C in temperature range. Here, we demonstrate the reversibility, robustness, and efficiency of these programmable DNA thermometers by monitoring temperature change inside individual wells during polymerase chain reactions. We discuss the potential applications of these programmable DNA thermoswitches in various nanotechnology fields including cell imaging, nanofluidics, nanomedecine, nanoelectronics, nanomaterial, and synthetic biology. PMID:27058370

  14. 173例系统性红斑狼疮患者ANA、ENA和dsDNA抗体的联合检测分析%Analysis of combined dectection of ANA, ENA and dsDNA in 173 Systemic Lupus Erythematosus patients

    Institute of Scientific and Technical Information of China (English)

    吴希国; 刘秀珍; 马越

    2012-01-01

    Objective:To evaluate the diagnostic valve of combined detection of ANA, ENA and dsDNA in systemic lupus enythmatosus. Methods; Serum was collected from 173 patients. ANA as well as dsDNA was tested by indirect immunofluorescent method, and ENA was tested by western blot. Results; The detection rates of the three antibodies in female SLE patients were higher than those of male patients. In the serum of patients,the detection rates of ANA, ASA( anti - sm antibodies) and dsDNA were 94. 8% , 32. 2% and 45. 1% respectively. Conclusion; There were significant differences in the sensitivity of ANA, ENA and dsDNA in systemic lupus erythematosus. Combined detection of ANA,ENA and dsDNA autoantibody had the diagnostic value to SLE.%目的:探讨ANA、ENA和dsDNA抗体联合检测在系统性红斑狼疮诊断中的意义.方法:收集173例患者血清,采用间接免疫荧光法检测ANA和dsDNA,用免疫印迹法测定ENA.结果:女性患者三种抗体同时阳性的检出率明显高于男性;患者血清中ANA阳性率为94.8%,抗dsDNA抗体为45.1%,抗Sm抗体为32.2%.结论:ANA、ENA和dsDNA三类抗体在SLE中的敏感性有显著的差异.这三类抗体的联合检测对SLE具有诊断意义.

  15. Impaired NFAT and NFκB activation are involved in suppression of CD40 ligand expression by Δ{sup 9}-tetrahydrocannabinol in human CD4{sup +} T cells

    Energy Technology Data Exchange (ETDEWEB)

    Ngaotepprutaram, Thitirat [Department of Pharmacology and Toxicology, Michigan State University (United States); Center for Integrative Toxicology, Michigan State University (United States); Kaplan, Barbara L.F. [Department of Pharmacology and Toxicology, Michigan State University (United States); Center for Integrative Toxicology, Michigan State University (United States); Neuroscience Program, Michigan State University (United States); Kaminski, Norbert E., E-mail: kamins11@msu.edu [Department of Pharmacology and Toxicology, Michigan State University (United States); Center for Integrative Toxicology, Michigan State University (United States)

    2013-11-15

    We have previously reported that Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), the main psychoactive cannabinoid in marijuana, suppresses CD40 ligand (CD40L) expression by activated mouse CD4{sup +} T cells. CD40L is involved in pathogenesis of many autoimmune and inflammatory diseases. In the present study, we investigated the molecular mechanism of Δ{sup 9}-THC-mediated suppression of CD40L expression using peripheral blood human T cells. Pretreatment with Δ{sup 9}-THC attenuated CD40L expression in human CD4{sup +} T cells activated by anti-CD3/CD28 at both the protein and mRNA level, as determined by flow cytometry and quantitative real-time PCR, respectively. Electrophoretic mobility shift assays revealed that Δ{sup 9}-THC suppressed the DNA-binding activity of both NFAT and NFκB to their respective response elements within the CD40L promoter. An assessment of the effect of Δ{sup 9}-THC on proximal T cell-receptor (TCR) signaling induced by anti-CD3/CD28 showed significant impairment in the rise of intracellular calcium, but no significant effect on the phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β. Collectively, these findings identify perturbation of the calcium-NFAT and NFκB signaling cascade as a key mechanistic event by which Δ{sup 9}-THC suppresses human T cell function. - Highlights: • Δ{sup 9}-THC attenuated CD40L expression in activated human CD4+ T cells. • Δ{sup 9}-THC suppressed DNA-binding activity of NFAT and NFκB. • Δ{sup 9}-THC impaired elevation of intracellular Ca2+. • Δ{sup 9}-THC did not affect phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β.

  16. Impaired NFAT and NFκB activation are involved in suppression of CD40 ligand expression by Δ9-tetrahydrocannabinol in human CD4+ T cells

    International Nuclear Information System (INIS)

    We have previously reported that Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive cannabinoid in marijuana, suppresses CD40 ligand (CD40L) expression by activated mouse CD4+ T cells. CD40L is involved in pathogenesis of many autoimmune and inflammatory diseases. In the present study, we investigated the molecular mechanism of Δ9-THC-mediated suppression of CD40L expression using peripheral blood human T cells. Pretreatment with Δ9-THC attenuated CD40L expression in human CD4+ T cells activated by anti-CD3/CD28 at both the protein and mRNA level, as determined by flow cytometry and quantitative real-time PCR, respectively. Electrophoretic mobility shift assays revealed that Δ9-THC suppressed the DNA-binding activity of both NFAT and NFκB to their respective response elements within the CD40L promoter. An assessment of the effect of Δ9-THC on proximal T cell-receptor (TCR) signaling induced by anti-CD3/CD28 showed significant impairment in the rise of intracellular calcium, but no significant effect on the phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β. Collectively, these findings identify perturbation of the calcium-NFAT and NFκB signaling cascade as a key mechanistic event by which Δ9-THC suppresses human T cell function. - Highlights: • Δ9-THC attenuated CD40L expression in activated human CD4+ T cells. • Δ9-THC suppressed DNA-binding activity of NFAT and NFκB. • Δ9-THC impaired elevation of intracellular Ca2+. • Δ9-THC did not affect phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β

  17. What Is Mitochondrial DNA?

    Science.gov (United States)

    ... DNA What is mitochondrial DNA? What is mitochondrial DNA? Although most DNA is packaged in chromosomes within ... proteins. For more information about mitochondria and mitochondrial DNA: Molecular Expressions, a web site from the Florida ...

  18. DNA and RNA sensor

    Institute of Scientific and Technical Information of China (English)

    LIU; Tao; LIN; Lin; ZHAO; Hong; JIANG; Long

    2005-01-01

    This review summarizes recent advances in DNA sensor. Major areas of DNA sensor covered in this review include immobilization methods of DNA, general techniques of DNA detection and application of nanoparticles in DNA sensor.

  19. Tetrahedron-structured DNA and functional oligonucleotide for construction of an electrochemical DNA-based biosensor.

    Science.gov (United States)

    Bu, Nan-Nan; Tang, Chun-Xia; He, Xi-Wen; Yin, Xue-Bo

    2011-07-21

    Tetrahedron-structured DNA (ts-DNA) in combination with a functionalized oligonucleotide was used to develop a "turn-on" biosensor for Hg(2+) ions. The ts-DNA provided an improved sensitivity and was used to block the active sites.

  20. DNA vaccines

    Science.gov (United States)

    Gregersen, Jens-Peter

    2001-12-01

    Immunization by genes encoding immunogens, rather than with the immunogen itself, has opened up new possibilities for vaccine research and development and offers chances for new applications and indications for future vaccines. The underlying mechanisms of antigen processing, immune presentation and regulation of immune responses raise high expectations for new and more effective prophylactic or therapeutic vaccines, particularly for vaccines against chronic or persistent infectious diseases and tumors. Our current knowledge and experience of DNA vaccination is summarized and critically reviewed with particular attention to basic immunological mechanisms, the construction of plasmids, screening for protective immunogens to be encoded by these plasmids, modes of application, pharmacokinetics, safety and immunotoxicological aspects. DNA vaccines have the potential to accelerate the research phase of new vaccines and to improve the chances of success, since finding new immunogens with the desired properties is at least technically less demanding than for conventional vaccines. However, on the way to innovative vaccine products, several hurdles have to be overcome. The efficacy of DNA vaccines in humans appears to be much less than indicated by early studies in mice. Open questions remain concerning the persistence and distribution of inoculated plasmid DNA in vivo, its potential to express antigens inappropriately, or the potentially deleterious ability to insert genes into the host cell's genome. Furthermore, the possibility of inducing immunotolerance or autoimmune diseases also needs to be investigated more thoroughly, in order to arrive at a well-founded consensus, which justifies the widespread application of DNA vaccines in a healthy population.

  1. DNA nanotechnology

    Directory of Open Access Journals (Sweden)

    Nadrian C Seeman

    2003-01-01

    We are all aware that the DNA found in cells is a double helix consisting of two antiparallel strands held together by specific hydrogen-bonded base pairs; adenine (A always pairs with thymine (T, and guanine (G always pairs with cytosine (C. The specificity of this base pairing and the ability to ensure that it occurs in this fashion (and not some other1 is key to the use of DNA in materials applications. The double helical arrangement of the two molecules leads to a linear helix axis, linear not in the geometrical sense of being a straight line, but in the topological sense of being unbranched. Genetic engineers discovered in the 1970s how to splice together pieces of DNA to add new genes to DNA molecules2, and synthetic chemists worked out convenient syntheses for short pieces of DNA (up to ∼100–150 units in the 1980s3. Regardless of the impact of these technologies on biological systems, hooking together linear molecules leads only to longer linear molecules, with circles, knots, and catenanes perhaps resulting from time to time.

  2. Structural organization of DNA in chlorella viruses.

    Directory of Open Access Journals (Sweden)

    Timo Wulfmeyer

    Full Text Available Chlorella viruses have icosahedral capsids with an internal membrane enclosing their large dsDNA genomes and associated proteins. Their genomes are packaged in the particles with a predicted DNA density of ca. 0.2 bp nm(-3. Occasionally infection of an algal cell by an individual particle fails and the viral DNA is dynamically ejected from the capsid. This shows that the release of the DNA generates a force, which can aid in the transfer of the genome into the host in a successful infection. Imaging of ejected viral DNA indicates that it is intimately associated with proteins in a periodic fashion. The bulk of the protein particles detected by atomic force microscopy have a size of ∼60 kDa and two proteins (A278L and A282L of about this size are among 6 basic putative DNA binding proteins found in a proteomic analysis of DNA binding proteins packaged in the virion. A combination of fluorescence images of ejected DNA and a bioinformatics analysis of the DNA reveal periodic patterns in the viral DNA. The periodic distribution of GC rich regions in the genome provides potential binding sites for basic proteins. This DNA/protein aggregation could be responsible for the periodic concentration of fluorescently labeled DNA observed in ejected viral DNA. Collectively the data indicate that the large chlorella viruses have a DNA packaging strategy that differs from bacteriophages; it involves proteins and share similarities to that of chromatin structure in eukaryotes.

  3. DNA nanotechnology

    OpenAIRE

    Seeman, Nadrian C.

    2003-01-01

    Since Watson and Crick’s determination of its structure nearly 50 years ago, DNA has come to fill our lives in many areas, from genetic counseling to forensics, from genomics to gene therapy. These, and other ways in which DNA affects human activities, are related to its function as genetic material, not just our genetic material, but the genetic material of all living organisms. Here, we will ignore DNA’s biological role; rather, we will discuss how the properties that make it so successful ...

  4. Combined Targeted DNA Sequencing in Non-Small Cell Lung Cancer (NSCLC Using UNCseq and NGScopy, and RNA Sequencing Using UNCqeR for the Detection of Genetic Aberrations in NSCLC.

    Directory of Open Access Journals (Sweden)

    Xiaobei Zhao

    Full Text Available The recent FDA approval of the MiSeqDx platform provides a unique opportunity to develop targeted next generation sequencing (NGS panels for human disease, including cancer. We have developed a scalable, targeted panel-based assay termed UNCseq, which involves a NGS panel of over 200 cancer-associated genes and a standardized downstream bioinformatics pipeline for detection of single nucleotide variations (SNV as well as small insertions and deletions (indel. In addition, we developed a novel algorithm, NGScopy, designed for samples with sparse sequencing coverage to detect large-scale copy number variations (CNV, similar to human SNP Array 6.0 as well as small-scale intragenic CNV. Overall, we applied this assay to 100 snap-frozen lung cancer specimens lacking same-patient germline DNA (07-0120 tissue cohort and validated our results against Sanger sequencing, SNP Array, and our recently published integrated DNA-seq/RNA-seq assay, UNCqeR, where RNA-seq of same-patient tumor specimens confirmed SNV detected by DNA-seq, if RNA-seq coverage depth was adequate. In addition, we applied the UNCseq assay on an independent lung cancer tumor tissue collection with available same-patient germline DNA (11-1115 tissue cohort and confirmed mutations using assays performed in a CLIA-certified laboratory. We conclude that UNCseq can identify SNV, indel, and CNV in tumor specimens lacking germline DNA in a cost-efficient fashion.

  5. The application of combination detection of hepatitis B serological markers quantifi-cation and HBV DNA quantification in the diagnosis of hepatitis B virus infection%乙肝血清学标志物定量和HBV DNA定量联合检测在乙肝病毒感染诊断中的应用

    Institute of Scientific and Technical Information of China (English)

    吴洪秋; 张永良; 黄坚尧; 张汉运

    2016-01-01

    Objective To analyze the application value of combination detection in the diagnosis of hepatitis B virus ( HBV) infection by the quantitative detection of serological markers and HBV DNA of hepatitis B patients. Methods The serum samples of 120 hepatitis B patients from Jun. 2013 to Jun. 2015 were collected, and the ELISA qualitative test and RT-PCR were used to detect the contents of HBV serological markers ( HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc) and HBV DNA. Results The HBV DNA positive rate was 96. 15% in Ⅰ group; the HBV DNA positive rate was 48. 08% in Ⅱ group;the HBV DNA positive rate was 4. 76% inⅢgroup. The quantitative HBV DNA inⅠgroup was significantly higher than that in Ⅱ and Ⅲ groups ( P0. 05). Conclusion The combination detection of HBV serological markers and HBV DNA quantification plays a complementary role in the diagnosis of HBV infection, and has a more accurate diagnosis as well as can provide a more effective reference.%目的:分析乙肝患者的血清标志物和乙肝病毒( HBV) DNA联合检测在HBV感染诊断中的应用价值。方法以2013年6月-2015年6月在珠海市妇幼保健院就诊的120例乙肝患者血清样本作为研究资料,分别采用ELISA定性试验和实时荧光定量PCR( RT-PCR)检测HBV血清标志物( HBsAg、抗-HBs、HBeAg、抗-HBe、抗-HBc)和HBV DNA含量。结果Ⅰ组HBV DNA阳性率为96.15%;Ⅱ组HBV DNA阳性率为48.08%;Ⅲ组HBV DNA阳性率为4.76%。Ⅰ组HBV DNA定量显著高于Ⅱ、Ⅲ两组( P0.05)。结论乙肝血清标志物定量与HBV DNA定量联合检测在诊断HBV感染过程中起互相补充的作用,诊断结果更加准确,可提供更有效的参考依据。

  6. A ranking index for quality assessment of forensic DNA profiles forensic DNA profiles

    Directory of Open Access Journals (Sweden)

    Ansell Ricky

    2010-11-01

    Full Text Available Abstract Background Assessment of DNA profile quality is vital in forensic DNA analysis, both in order to determine the evidentiary value of DNA results and to compare the performance of different DNA analysis protocols. Generally the quality assessment is performed through manual examination of the DNA profiles based on empirical knowledge, or by comparing the intensities (allelic peak heights of the capillary electrophoresis electropherograms. Results We recently developed a ranking index for unbiased and quantitative quality assessment of forensic DNA profiles, the forensic DNA profile index (FI (Hedman et al. Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modeling of DNA profiles, Biotechniques 47 (2009 951-958. FI uses electropherogram data to combine the intensities of the allelic peaks with the balances within and between loci, using Principal Components Analysis. Here we present the construction of FI. We explain the mathematical and statistical methodologies used and present details about the applied data reduction method. Thereby we show how to adapt the ranking index for any Short Tandem Repeat-based forensic DNA typing system through validation against a manual grading scale and calibration against a specific set of DNA profiles. Conclusions The developed tool provides unbiased quality assessment of forensic DNA profiles. It can be applied for any DNA profiling system based on Short Tandem Repeat markers. Apart from crime related DNA analysis, FI can therefore be used as a quality tool in paternal or familial testing as well as in disaster victim identification.

  7. HIV DNA Vaccine: Stepwise Improvements Make a Difference

    Directory of Open Access Journals (Sweden)

    Barbara K. Felber

    2014-05-01

    Full Text Available Inefficient DNA delivery methods and low expression of plasmid DNA have been major obstacles for the use of plasmid DNA as vaccine for HIV/AIDS. This review describes successful efforts to improve DNA vaccine methodology over the past ~30 years. DNA vaccination, either alone or in combination with other methods, has the potential to be a rapid, safe, and effective vaccine platform against AIDS. Recent clinical trials suggest the feasibility of its translation to the clinic.

  8. Osteogenesis and vascularization of bone marrow mesenchymal stem cells transfected by pcDNA3/hVEGF165 combined with freeze-dried cancellous bone in vivo%脂质体介导pcDNA3/hVEGF165转染骨髓基质干细胞复合冻干骨的体内成骨和血管化

    Institute of Scientific and Technical Information of China (English)

    张鹏; 董玲; 杨连甲

    2011-01-01

    BACKGROUND: Previous studies have shown that vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) can improve the implant livability and growth.OBJECTIVE: To study the effect of freeze-dried cancellous bone on the osteogenesis and vascularization of bone marrow stem cells transfected with pcDNA3/hVEGF165 in vivo.METHODS: The rabbit bone marrow stem cells, which were transfected with pcDNA3/hVEGF165 by liposome mediated method and then adhered to freeze-dried cancellous bone, were implanted in the muscle pouches of rabbits. The rabbits were divided into three groups: freeze-dried cancellous bone group (A group), freeze-dried cancellous bone combined with bone marrow stem cells group (B group), freeze-dried cancellous bone combined with bone marrow stem cells transfected with VEGF group (C group).RESULTS AND CONCLUSION: At the 8th week after implantation, it was found, compared with A group and B group, C group grew a large number of osteoblasts, osteoclasts and cartilage, and the number of vessels in C group was more than that in A or B group. The osteogenesis of the freeze-dried cancellous bone combined with bone marrow stem cells transfected with VEGF using pcDNA3/hVEGF165 by liposome mediated method is better than the freeze-dried cancellous bone or freeze-dried cancellous bone combined with bone marrow stem cells.%背景:以往的研究表明血管内皮生长因子、碱性成纤维细胞生长因子可以促进移植物的存活和体内生长.目的:观察脂质体介导的pcDNA3/hVEGF165转染骨髓基质干细胞后复合冻干松质骨在体内的成骨和血管化效果.方法:取同种异体新西兰大白兔的耾骨和股骨制备冻干骨,用脂质体将血管内皮生长因子转染入体外培养扩增新西兰大白兔骨髓基质干细胞中,使其附着于同种异体冻干松质骨.将新西兰大白兔分为3组,于兔竖脊肌分别植入单纯冻干骨、单纯骨髓间充质干细胞复合冻干骨组、转染

  9. Analysis of the Framework of Microsoft DNA

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Microsoft DNA(Distributed interNet Application) has p rovided a effective framework of combination PC with the Internet perfectly; In addition, this open framework hase also given the integration of multilingual co mponents a strong managing platform. This article has deeply analysed the main technologys of Microsoft DNA, involve COM basic technology, framework of DNA, Microfost UDA(Universal Data Access),com ponent management with COM+, asynchronous communication with MSMQ(M icrosoft Message Queue). In...

  10. Diatom DNA as historical information

    Science.gov (United States)

    Stoof, K. R.; Epp, L. S.; Trauth, M. H.; Tiedemann, R.

    2009-04-01

    This survey is about the reconstruction of paleo environmental conditions in East Africa using recent and historic lakes sediments of shallow lakes in Kenya. This interdisciplinary approach combines the molecular analysis of ancient diatom DNA and the morphological analysis of diatom assemblages of short sediment cores to reveal the potential of molecular genetics in geosciences. This study comprises the investigation of two short sediment cores from Lake Naivasha (Kenya) taken in 2007, covering a stretch of ca. 80 years. Several sediment slices were analysed by molecular methods that concludes in species identification based on DNA fragments. Therefore total DNA was extracted and applied to a diatom DNA specific PCR amplifying a gene fragment that is frequently used for species identification. After cloning PCR products, clones were sequenced, sequences from different diatom species were analysed. As a comparison permanent slides were prepared for each sediment sample for counting diatom valves. The results suggest that DNA damage, perhaps species depended, and specific factors of PCR contribute to an overhang of two dominant diatom species (Aulacoseira ambigua and Aulacoseira granulata) represented by the molecular approach whereas rare species seen by light microscopy are not yet identifiable with molecular methods. Moreover changes in diatom assemblages and the reconstructed conductivity indicate a decrease of lake level around 1940. This shift seems to be also represented in the molecular approach as a decrease in the amount of diverse diatom DNA sequences found, what is probably caused by worse DNA preservation conditions at that time.

  11. Detection of Non-Amplified Genomic DNA

    CERN Document Server

    Corradini, Roberto

    2012-01-01

    This book offers a state-of-the-art overview on non amplified DNA detection methods and provides chemists, biochemists, biotechnologists and material scientists with an introduction to these methods. In fact all these fields have dedicated resources to the problem of nucleic acid detection, each contributing with their own specific methods and concepts. This book will explain the basic principles of the different non amplified DNA detection methods available, highlighting their respective advantages and limitations. The importance of non-amplified DNA sequencing technologies will be also discussed. Non-amplified DNA detection can be achieved by adopting different techniques. Such techniques have allowed the commercialization of innovative platforms for DNA detection that are expected to break into the DNA diagnostics market. The enhanced sensitivity required for the detection of non amplified genomic DNA has prompted new strategies that can achieve ultrasensitivity by combining specific materials with specifi...

  12. DIBER: protein, DNA or both?

    Science.gov (United States)

    Chojnowski, Grzegorz; Bochtler, Matthias

    2010-06-01

    The program DIBER (an acronym for DNA and FIBER) requires only native diffraction data to predict whether a crystal contains protein, B-form DNA or both. In standalone mode, the classification is based on the cube root of the reciprocal unit-cell volume and the largest local average of diffraction intensities at 3.4 A resolution. In combined mode, the Phaser rotation-function score (for the 3.4 A shell and a canonical B-DNA search model) is also taken into account. In standalone (combined) mode, DIBER classifies 87.4 +/- 0.2% (90.2 +/- 0.3%) of protein, 69.1 +/- 0.3% (78.8 +/- 0.3%) of protein-DNA and 92.7 +/- 0.2% (90.0 +/- 0.2%) of DNA crystals correctly. Reliable predictions with a correct classification rate above 80% are possible for 36.8 +/- 1.0% (60.2 +/- 0.4%) of the protein, 43.6 +/- 0.5% (59.8 +/- 0.3%) of the protein-DNA and 83.3 +/- 0.3% (82.6 +/- 0.4%) of the DNA structures. Surprisingly, selective use of the diffraction data in the 3.4 A shell improves the overall success rate of the combined-mode classification. An open-source CCP4/CCP4i-compatible version of DIBER is available from the authors' website at http://www.iimcb.gov.pl/diber and is subject to the GNU Public License. PMID:20516617

  13. DNA Microarrays

    Science.gov (United States)

    Nguyen, C.; Gidrol, X.

    Genomics has revolutionised biological and biomedical research. This revolution was predictable on the basis of its two driving forces: the ever increasing availability of genome sequences and the development of new technology able to exploit them. Up until now, technical limitations meant that molecular biology could only analyse one or two parameters per experiment, providing relatively little information compared with the great complexity of the systems under investigation. This gene by gene approach is inadequate to understand biological systems containing several thousand genes. It is essential to have an overall view of the DNA, RNA, and relevant proteins. A simple inventory of the genome is not sufficient to understand the functions of the genes, or indeed the way that cells and organisms work. For this purpose, functional studies based on whole genomes are needed. Among these new large-scale methods of molecular analysis, DNA microarrays provide a way of studying the genome and the transcriptome. The idea of integrating a large amount of data derived from a support with very small area has led biologists to call these chips, borrowing the term from the microelectronics industry. At the beginning of the 1990s, the development of DNA chips on nylon membranes [1, 2], then on glass [3] and silicon [4] supports, made it possible for the first time to carry out simultaneous measurements of the equilibrium concentration of all the messenger RNA (mRNA) or transcribed RNA in a cell. These microarrays offer a wide range of applications, in both fundamental and clinical research, providing a method for genome-wide characterisation of changes occurring within a cell or tissue, as for example in polymorphism studies, detection of mutations, and quantitative assays of gene copies. With regard to the transcriptome, it provides a way of characterising differentially expressed genes, profiling given biological states, and identifying regulatory channels.

  14. Synthesis and structure elucidation of new μ-oxamido-bridged dicopper(II) complex with in vitro anticancer activity: A combined study from experiment verification and docking calculation on DNA/protein-binding property.

    Science.gov (United States)

    Zhu, Ling; Zheng, Kang; Li, Yan-Tuan; Wu, Zhi-Yong; Yan, Cui-Wei

    2016-02-01

    A new oxamido-bridged dicopper(II) complex with formula of [Cu2(deap)(pic)2], where H2deap and pic represent N,N'-bis[3-(diethylamino)propyl]oxamide and picrate, respectively, was synthesized and characterized by elemental analyses, molar conductance measurements, IR and electronic spectral study, and single-crystal X-ray diffraction. The crystal structure analyses revealed that the two copper(II) atoms in the dicopper(II) complex are bridged by the trans-deap(2-) ligand with the distances of 5.2116(17)Å, and the coordination environment around the copper(II) atoms can be described as a square-planar geometry. Hydrogen bonding and π-π stacking interactions link the dicopper(II) complex into a three-dimensional infinite network. The DNA/protein-binding properties of the complex are investigated by molecular docking and experimental assays. The results indicate that the dicopper(II) complex can interact with HS-DNA in the mode of intercalation and effectively quench the intrinsic fluorescence of protein BSA by 1:1 binding with the most possible binding site in the proximity of Trp134. The in vitro anticancer activities suggest that the complex is active against the selected tumor cell lines, and IC50 values for SMMC-7721 and HepG2 are lower than cisplatin. The effects of the electron density distribution of the terminal ligand and the chelate ring arrangement around copper(II) ions bridged by symmetric N,N'-bis(substituted)oxamides on DNA/BSA-binding ability and in vitro anticancer activity are preliminarily discussed.

  15. Diagnostic value of cervical cytological examination, HPV-DNA test combined with colposcopy examination in cervical lesions diagnosis%宫颈细胞学检查、HPV-DNA检测配合阴道镜检查对子宫颈病变的诊断价值

    Institute of Scientific and Technical Information of China (English)

    李玉欢

    2013-01-01

    目的 分析与探讨宫颈细胞学检查、HPV-DNA(人乳头瘤病毒基因检测)检测配合阴道镜检查对于子宫颈病变的诊断价值.方法 选取本院2010年8月至2012年8月期间收治的接受子宫颈病变筛查的妇女共1020例,对其实施宫颈细胞学检查和HPV-DNA配合阴道镜检查,检查结果与组织学金标准进行比对,以对比两种方法的准确性与诊断价值.结果 采取HPV-DNA配合阴道镜检查为异常结果的检出率较宫颈细胞学检查更高,差异具有统计学意义(P<0.05),前者检出异常共64例,后者检出异常共34例.结论 宫颈细胞学检查、HPV-DNA检测以及阴道镜检查对于子宫颈病变均有一定的诊断价值,而采用HPV-DNA检测配合阴道镜检查能够帮助医生较早地发现患者的宫颈癌前病变,为患者的治疗方案的选择提供科学依据.%Objective To analyze and discuss the diagnostic value of cervical cytological examination,HPV-DNA test combined with colposcopy examination in diagnosis of cervical lesions.Methods From August 2010 to August 2012 in our hospital a total of 1020 cases of women who underwent cervical diseases screening,were implemented with cervical cytological examination and HPV-DNA combined with colposcopy examination,the correlated pathological results were compared with the gold standard in accuracy and diagnostic value of two methods.Results The HPV-DNA abnormal results detection rate was higher than that of cervical cytological examination combined with colposcopy examination,with statistically significant difference,former found 64 abnormal cases,the latter examination found 34 cases of abnormal.Conclusion Cervical cytological examination,HPV-DNA testing combined with colposcopy examination for cervical lesions has certain diagnostic value,and the combined use of HPV-DNA testing and colposcopy examination could help doctors find early precancerous lesion of cervical cancer,and provide scientific basis for the

  16. Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA and lengthen linear DNA

    Energy Technology Data Exchange (ETDEWEB)

    Verebová, Valéria [Institute of Biophysics, University of Veterinary Medicine and Pharmacy, Komenského 73, 041 81 Košice (Slovakia); Adamcik, Jozef [Food and Soft Materials Science, Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 9, CH-8092 Zürich (Switzerland); Danko, Patrik; Podhradský, Dušan [Department of Biochemistry, Institute of Chemistry, Faculty of Sciences, P.J. Šafárik University, Moyzesova 11, 041 54 Košice (Slovakia); Miškovský, Pavol [Department of Biophysics, Faculty of Sciences, P.J. Šafárik University, Jesenná 5, 041 54 Košice (Slovakia); Center for Interdisciplinary Biosciences, Faculty of Sciences, P.J. Šafárik University, Jesenná 5, 041 54 Košice (Slovakia); Staničová, Jana, E-mail: jana.stanicova@uvlf.sk [Institute of Biophysics, University of Veterinary Medicine and Pharmacy, Komenského 73, 041 81 Košice (Slovakia)

    2014-01-31

    Highlights: • Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA. • Anthraquinones quinizarin and danthron lengthen linear DNA. • Anthraquinones quinizarin and danthron possess middle binding affinity to DNA. • Anthraquinones quinizarin and danthron interact with DNA by intercalating mode. - Abstract: The intercalating drugs possess a planar aromatic chromophore unit by which they insert between DNA bases causing the distortion of classical B-DNA form. The planar tricyclic structure of anthraquinones belongs to the group of chromophore units and enables anthraquinones to bind to DNA by intercalating mode. The interactions of simple derivatives of anthraquinone, quinizarin (1,4-dihydroxyanthraquinone) and danthron (1,8-dihydroxyanthraquinone), with negatively supercoiled and linear DNA were investigated using a combination of the electrophoretic methods, fluorescence spectrophotometry and single molecule technique an atomic force microscopy. The detection of the topological change of negatively supercoiled plasmid DNA, unwinding of negatively supercoiled DNA, corresponding to appearance of DNA topoisomers with the low superhelicity and an increase of the contour length of linear DNA in the presence of quinizarin and danthron indicate the binding of both anthraquinones to DNA by intercalating mode.

  17. Analysis of differentially expressed tumor-related genes in Peutz-Jeghers syndrome combined with colorectal carcinoma with cDNA microarrays%对比分析黑斑息肉综合征合并大肠癌组织肿瘤基因

    Institute of Scientific and Technical Information of China (English)

    Xiaosan Zhu; Qiaofang Sang; Xiaojuan Zhan; Yichen Dai; Qingzhen Nan; Zhangxin Chen; Junpei Xie; Wei Zeng; Yuka Fu; Yuanyuan Lin; Qingna Lian

    2011-01-01

    Objective: The aim of the study was to screen the differentially expressed genes of Peutz-Jeghers syndrome (PJS) and colorectal carcinoma (CRC). Methods: This study used cDNA microarray to comparatively analyze the gene expression profiles of 4 cases of PJS combined with colorectal adenocarcinoma vs. normal mucosae. The cDNA microarray contained 8064 human genes, and then using RT-PCR to test three genes of all. Results: The experimental data showed that fourteen genes were differentially expressed, which were up-regulated in PJS. Fifty-one genes expressions were altered in CRCs, of which 32 were up-regulated, as compared to the normal mucosae. In addition, 5 genes were similarly altered in both PJS and CRCs. RT-PCR analyses confirmed the cDNA microarray data for three of those genes: LATS2, APC and MADH4. Conclusion: LCN2, USP4, GRO3, HYAL1 and APC - these differentially expressed genes likely represent biomarkers for early detection of CRC and may be potential therapeutic targets.

  18. DNA detection using recombination proteins.

    Directory of Open Access Journals (Sweden)

    Olaf Piepenburg

    2006-07-01

    Full Text Available DNA amplification is essential to most nucleic acid testing strategies, but established techniques require sophisticated equipment or complex experimental procedures, and their uptake outside specialised laboratories has been limited. Our novel approach, recombinase polymerase amplification (RPA, couples isothermal recombinase-driven primer targeting of template material with strand-displacement DNA synthesis. It achieves exponential amplification with no need for pretreatment of sample DNA. Reactions are sensitive, specific, and rapid and operate at constant low temperature. We have also developed a probe-based detection system. Key aspects of the combined RPA amplification/detection process are illustrated by a test for the pathogen methicillin-resistant Staphylococcus aureus. The technology proves to be sensitive to fewer than ten copies of genomic DNA. Furthermore, products can be detected in a simple sandwich assay, thereby establishing an instrument-free DNA testing system. This unique combination of properties is a significant advance in the development of portable and widely accessible nucleic acid-based tests.

  19. Pig transgenesis by Sleeping Beauty DNA transposition

    DEFF Research Database (Denmark)

    Jakobsen, Jannik E.; Li, Juan; Kragh, Peter M.;

    2011-01-01

    disease models. In this report, we present transgenic pigs created by Sleeping Beauty DNA transposition in primary porcine fibroblasts in combination with somatic cell nuclear transfer by handmade cloning. Göttingen minipigs expressing green fluorescent protein are produced by transgenesis with DNA...... plasmid DNA. Our findings illustrate critical issues related to DNA transposon-directed transgenesis, including coincidental plasmid insertion and relatively low Sleeping Beauty transposition activity in porcine fibroblasts, but also provide a platform for future development of porcine disease models...... using the Sleeping Beauty gene insertion technology....

  20. DNA damage and repair in plants

    International Nuclear Information System (INIS)

    The biological impact of any DNA damaging agent is a combined function of the chemical nature of the induced lesions and the efficiency and accuracy of their repair. Although much has been learned frommicrobes and mammals about both the repair of DNA damage and the biological effects of the persistence of these lesions, much remains to be learned about the mechanism and tissue-specificity of repair in plants. This review focuses on recent work on the induction and repair of DNA damage in higher plants, with special emphasis on UV-induced DNA damage products. (author)

  1. Clinical trial in healthy malaria-naïve adults to evaluate the safety, tolerability, immunogenicity and efficacy of MuStDO5, a five-gene, sporozoite/hepatic stage Plasmodium falciparum DNA vaccine combined with escalating dose human GM-CSF DNA

    Science.gov (United States)

    Richie, Thomas L.; Charoenvit, Yupin; Wang, Ruobing; Epstein, Judith E.; Hedstrom, Richard C.; Kumar, Sanjai; Luke, Thomas C.; Freilich, Daniel A.; Aguiar, Joao C.; Sacci, Jr., John B.; Sedegah, Martha; Nosek, Jr., Ronald A.; De La Vega, Patricia; Berzins, Mara P.; Majam, Victoria F.; Abot, Esteban N.; Ganeshan, Harini; Richie, Nancy O.; Banania, Jo Glenna; Baraceros, Maria Fe B.; Geter, Tanya G.; Mere, Robin; Bebris, Lolita; Limbach, Keith; Hickey, Bradley W.; Lanar, David E.; Ng, Jennifer; Shi, Meng; Hobart, Peter M.; Norman, Jon A.; Soisson, Lorraine A.; Hollingdale, Michael R.; Rogers, William O.; Doolan, Denise L.; Hoffman, Stephen L.

    2012-01-01

    When introduced in the 1990s, immunization with DNA plasmids was considered potentially revolutionary for vaccine development, particularly for vaccines intended to induce protective CD8 T cell responses against multiple antigens. We conducted, in 1997−1998, the first clinical trial in healthy humans of a DNA vaccine, a single plasmid encoding Plasmodium falciparum circumsporozoite protein (PfCSP), as an initial step toward developing a multi-antigen malaria vaccine targeting the liver stages of the parasite. As the next step, we conducted in 2000–2001 a clinical trial of a five-plasmid mixture called MuStDO5 encoding pre-erythrocytic antigens PfCSP, PfSSP2/TRAP, PfEXP1, PfLSA1 and PfLSA3. Thirty-two, malaria-naïve, adult volunteers were enrolled sequentially into four cohorts receiving a mixture of 500 μg of each plasmid plus escalating doses (0, 20, 100 or 500 μg) of a sixth plasmid encoding human granulocyte macrophage-colony stimulating factor (hGM-CSF). Three doses of each formulation were administered intramuscularly by needle-less jet injection at 0, 4 and 8 weeks, and each cohort had controlled human malaria infection administered by five mosquito bites 18 d later. The vaccine was safe and well-tolerated, inducing moderate antigen-specific, MHC-restricted T cell interferon-γ responses but no antibodies. Although no volunteers were protected, T cell responses were boosted post malaria challenge. This trial demonstrated the MuStDO5 DNA and hGM-CSF plasmids to be safe and modestly immunogenic for T cell responses. It also laid the foundation for priming with DNA plasmids and boosting with recombinant viruses, an approach known for nearly 15 y to enhance the immunogenicity and protective efficacy of DNA vaccines. PMID:23151451

  2. Research on the source of varieties and high performance liquid chromatography of Brazilian herb BARDANA based on a combined analysis of DNA barcoding-origin-morphology%基于 DNA 条形码-产地-形态分析联用的巴西草药 BARDANA(牛蒡)的品种鉴定及高效液相色谱研究

    Institute of Scientific and Technical Information of China (English)

    余春霞; 顾选; 李妍芃; 肖瑶; 赵百孝; 刘春生; 马长华; 韩丽

    2015-01-01

    Objective To trace the source and study on the content of arctiin of Brazilian herb BARDANA for varieties systematics and pharmacological analysis. Methods The source of varieties of Brazilian herb BARDANA was identified by a technology combined DNA barcoding, plant geographical origin and morphological features , and the content of arctiin was analyzed by HPLC. Results DNA barcoding preliminarily identified BARDANA belongs to the stretch of Arctium of Compositae with a similarity level of 99% . Geographical and morphological verification further confirmed the aforementioned finding. The content of arctiin in the Brazilian was 0. 281% ,which was significantly lower than the Chinese arctiin (3. 72% ). Conclusion Brazilian herb BARDANA derives from Arctium lappa L. , and the content of arctiin was very low.%目的:对巴西草药牛蒡的品种进行鉴定,同时研究其中的牛蒡苷含量,为其品种整理和药理研究提供依据。方法利用 DNA 条形码—产地—形态分析联用技术鉴定巴西草药牛蒡的基原,高效液相色谱法测定其牛蒡苷含量。结果 DNA 条形码初步鉴定巴西草药牛蒡为菊科牛蒡属植物,且与牛蒡达到最高相似度99%,产地及形态分析进一步确证其为牛蒡的地上部分;巴西牛蒡草中牛蒡苷平均含量为0.281%,明显低于中国牛蒡草牛蒡苷平均含量3.72%。结论巴西草药牛蒡来源于牛蒡 Arctium lappa L.的干燥地上部分,其牛蒡苷含量极低。

  3. 基于 DNA 条形码-产地-形态分析联用的巴西草药 BARDANA(牛蒡)的品种鉴定及高效液相色谱研究%Research on the source of varieties and high performance liquid chromatography of Brazilian herb BARDANA based on a combined analysis of DNA barcoding-origin-morphology

    Institute of Scientific and Technical Information of China (English)

    余春霞; 顾选; 李妍芃; 肖瑶; 赵百孝; 刘春生; 马长华; 韩丽

    2015-01-01

    Objective To trace the source and study on the content of arctiin of Brazilian herb BARDANA for varieties systematics and pharmacological analysis. Methods The source of varieties of Brazilian herb BARDANA was identified by a technology combined DNA barcoding, plant geographical origin and morphological features , and the content of arctiin was analyzed by HPLC. Results DNA barcoding preliminarily identified BARDANA belongs to the stretch of Arctium of Compositae with a similarity level of 99% . Geographical and morphological verification further confirmed the aforementioned finding. The content of arctiin in the Brazilian was 0. 281% ,which was significantly lower than the Chinese arctiin (3. 72% ). Conclusion Brazilian herb BARDANA derives from Arctium lappa L. , and the content of arctiin was very low.%目的:对巴西草药牛蒡的品种进行鉴定,同时研究其中的牛蒡苷含量,为其品种整理和药理研究提供依据。方法利用 DNA 条形码—产地—形态分析联用技术鉴定巴西草药牛蒡的基原,高效液相色谱法测定其牛蒡苷含量。结果 DNA 条形码初步鉴定巴西草药牛蒡为菊科牛蒡属植物,且与牛蒡达到最高相似度99%,产地及形态分析进一步确证其为牛蒡的地上部分;巴西牛蒡草中牛蒡苷平均含量为0.281%,明显低于中国牛蒡草牛蒡苷平均含量3.72%。结论巴西草药牛蒡来源于牛蒡 Arctium lappa L.的干燥地上部分,其牛蒡苷含量极低。

  4. Combined detection of sputum smears ,sputum TB-DNA and serum anti-PPD-IgG in diagnosis of tuberculosis%痰涂片和TB-DNA及血清抗PPD-IgG在肺结核感染诊断中的联合应用

    Institute of Scientific and Technical Information of China (English)

    张梅

    2016-01-01

    目的 探讨痰 TB-DNA 定性与血清抗结核抗体及痰抗酸染色检测方法在肺结核感染诊断中的联合应用价值.方法 以2013年1月至2015年6月在清远市人民医院就诊的肺结核患者278例为研究对象.另择性别、年龄匹配的同期入院非肺结核病患者121例为对照组.现通过收集该肺结核患者的TB-DNA定性和血清抗结核抗体及抗酸染色的检测结果进行分析 ,探讨痰TB-DNA定性与血清抗结核抗体检测及抗酸染色在肺结核感染诊断中的联合应用.结果 结核病组中抗酸染色灵敏度为32 .01% ;血清抗结核抗体灵敏度为51 .44% ;TB-DNA定性灵敏度为48 .56% ;TB-DNA定性和血清抗结核抗体联合检出率为67 .63% ;TB-DNA定性和抗酸染色联合检出率为54 .68% ;血清抗结核抗体和抗酸染色联合检出率为57 .55% ;三者联合检出率为68 .71% .结论 血清抗结核抗体检测对肺结核的诊断简单、有效 ,灵敏度优于TB-DNA定性检测及抗酸染色 ,但其特异度比较低.细菌学诊断是结核病诊断的"金标准" ,但是痰涂片寻找抗酸杆菌的阳性率低 ,而 TB-DNA定性检测快速又准确.血清抗结核抗体及痰抗酸染色及痰TB-DNA定性检测联合应用于肺结核诊断效果更佳.%Objective To explore the value of combined application of qualitative detection of TB-DNA serum anti-PPD-IgG and acid-fast staining methods in the diagnosis of tuberculosis infection .Methods Totally 278 pulmonary tuberculosis patients and 121 non-pulmonary tuberculosis patients were collected from Qingyuan people′s hospital during the period from January 2013 to June 2015 .Tuberculosis in patients with TB-DNA qualitative and serum anti-PPD-IgG and acid-fast staining test results was analyzed . Sputum TB-DNA qualitative and serum anti-PPD-IgG detection and joint application of acid-fast staining in the diagnosis of tuber-culosis infection .Results Sensitivities of acid-fast staining ,TB-DNA and serum anti

  5. A Phase I Double Blind, Placebo-Controlled, Randomized Study of the Safety and Immunogenicity of Electroporated HIV DNA with or without Interleukin 12 in Prime-Boost Combinations with an Ad35 HIV Vaccine in Healthy HIV-Seronegative African Adults.

    Directory of Open Access Journals (Sweden)

    Juliet Mpendo

    Full Text Available Strategies to enhance the immunogenicity of DNA vaccines in humans include i co-administration of molecular adjuvants, ii intramuscular administration followed by in vivo electroporation (IM/EP and/or iii boosting with a different vaccine. Combining these strategies provided protection of macaques challenged with SIV; this clinical trial was designed to mimic the vaccine regimen in the SIV study.Seventy five healthy, HIV-seronegative adults were enrolled into a phase 1, randomized, double-blind, placebo-controlled trial. Multi-antigenic HIV (HIVMAG plasmid DNA (pDNA vaccine alone or co-administered with pDNA encoding human Interleukin 12 (IL-12 (GENEVAX IL-12 given by IM/EP using the TriGrid Delivery System was tested in different prime-boost regimens with recombinant Ad35 HIV vaccine given IM.All local reactions but one were mild or moderate. Systemic reactions and unsolicited adverse events including laboratory abnormalities did not differ between vaccine and placebo recipients. No serious adverse events (SAEs were reported. T cell and antibody response rates after HIVMAG (x3 prime-Ad35 (x1 boost were independent of IL-12, while the magnitude of interferon gamma (IFN-γ ELISPOT responses was highest after HIVMAG (x3 without IL-12. The quality and phenotype of T cell responses shown by intracellular cytokine staining (ICS were similar between groups. Inhibition of HIV replication by autologous T cells was demonstrated after HIVMAG (x3 prime and was boosted after Ad35. HIV specific antibodies were detected only after Ad35 boost, although there was a priming effect with 3 doses of HIVMAG with or without IL-12. No anti-IL-12 antibodies were detected.The vaccines were safe, well tolerated and moderately immunogenic. Repeated administration IM/EP was well accepted. An adjuvant effect of co-administered plasmid IL-12 was not detected.ClinicalTrials.gov NCT01496989.

  6. Protection against SHIV-KB9 Infection by Combining rDNA and rFPV Vaccines Based on HIV Multiepitope and p24 Protein in Chinese Rhesus Macaques

    Directory of Open Access Journals (Sweden)

    Chang Li

    2012-01-01

    Full Text Available Developing an effective vaccine against HIV infection remains an urgent goal. We used a DNA prime/fowlpox virus boost regimen to immunize Chinese rhesus macaques. The animals were challenged intramuscularly with pathogenic molecularly cloned SHIV-KB9. Immunogenicity and protective efficacy of vaccines were investigated by measuring IFN-γ levels, monitoring HIV-specific binding antibodies, examining viral load, and analyzing CD4/CD8 ratio. Results show that, upon challenge, the vaccine group can induce a strong immune response in the body, represented by increased expression of IFN-γ, slow and steady elevated antibody production, reduced peak value of acute viral load, and increase in the average CD4/CD8 ratio. The current research suggests that rapid reaction speed, appropriate response strength, and long-lasting immune response time may be key protection factors for AIDS vaccine. The present study contributes significantly to AIDS vaccine and preclinical research.

  7. 乙肝不同血清模式及乙肝DNA定量与前S1抗原联合检测的临床意义%Clinical values of combined analysis on serological patterns, virus DNA titer and pre-S1 antigen for hepatitis B

    Institute of Scientific and Technical Information of China (English)

    王凤敏; 李志

    2016-01-01

    目的 探讨乙型肝炎患者不同的血清学模式、乙肝病毒DNA(HBV-DNA)与乙肝前S1抗原(Pre-S1 Ag)联合检测的临床意义.方法 采用化学免疫发光法(CLIA)定量筛选339例乙肝血清标志物阳性血清,采用荧光定量聚合酶链反应法(FQ-PCR)检测HBV-DNA,采用酶联免疫吸附法(ELISA)检测Pre-S1 Ag.结果 乙肝不同血清模式下,HBV-DNA与Pre-S1 Ag检测结果比较差异无统计学意义(P>0.05).HBsAg、HBeAg、抗-HBc阳性组HBV-DNA检出率93.1%,Pre-S1Ag检出率86.1%.HBsAg、HBeAb、抗-HBc阳性组HBV-DNA检出率45.9%,Pre-S1 Ag检出率69.2%.HBsAg、抗-HBc阳性组HBV-DNA检出率61.0%,Pre-S1 Ag检出率72.9%.HBsAg、HBeAg阳性组HBV-DNA及Pre-S1 Ag检出率均为100%.以HBeAg阳性为对照HBV-DNA及Pre-S1 Ag检出率分别为87.3%和93.7%.HBV-DNA与Pre-S1 Ag检测结果比较差异有统计学意义(P>0.05).结论 乙肝五项、HBV-DNA、Pre-S1Ag联合检测能够对乙肝病毒的感染、复制程度做出准确的判断,为临床治疗方案的选择和疗效的观察提供可靠的依据.%Objective To discuss clinical values of the combined analysis on serological patterns, virus DNA titer and pre-S1 antigen for hepatitis B.Methods Serum samples from 339 patients with chemical luminescent immunoassay (CLIA) positive results were used in the study.The level of serum HBV-DNA was detected by fluorescent quantitative polymerase chain reaction (FQ-PCR).Pre-S1Ag was detected by ELISA.Results There was no significant differences in pre-S1 Ag or HBV-DNA with different serological patterns of hepatitis B (P > 0.05).The HBV-DNA positive rate of the HBsAg, HBeAg and Anti-HBc positive group was 93.1% while the pre-S1Ag positive rate was 86.1%.The HBV-DNA positive rate of the HB-sAg, HBeAb, anti-HBc positive group was 45.9% and the Pre-S1Ag positive rate was 69.2%.The HBV-DNA positive rate of the HBsAg, anti-HBc positive groups was 61.0%.The Pre-S1Ag positive rate was 72.9

  8. The Effects of Combined Antioxidant Supplementation on Antioxidant Capacity, DNA Single-Strand Breaks and Regulation of Insulin Growth Factor-1/IGF-Binding Protein 3 in the Ferret Model of Lung Cancer

    Science.gov (United States)

    Purpose: Insulin-like growth factor 1 (IGF-1) and its major binding protein, IGF binding protein 3 (IGFBP-3) are implicated in lung cancer and other malignancies. We have previously shown that the combination of three major antioxidants [beta-carotene (BC), alpha-tocopherol (AT) and ascorbic acid (...

  9. Sperm DNA oxidative damage and DNA adducts.

    Science.gov (United States)

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-12-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps=0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps=0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps=0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on sperm

  10. Synthesis of DNA

    Science.gov (United States)

    Mariella, Jr., Raymond P.

    2008-11-18

    A method of synthesizing a desired double-stranded DNA of a predetermined length and of a predetermined sequence. Preselected sequence segments that will complete the desired double-stranded DNA are determined. Preselected segment sequences of DNA that will be used to complete the desired double-stranded DNA are provided. The preselected segment sequences of DNA are assembled to produce the desired double-stranded DNA.

  11. DNA repair in human bronchial epithelial cells

    International Nuclear Information System (INIS)

    The purpose of this investigation was to compare the response of human cell types (bronchial epithelial cells and fibroblasts and skin fibroblasts) to various DNA damaging agents. Repair of DNA single strand breaks (SSB) induced by 5 krads of X-ray was similar for all cell types; approximately 90% of the DNA SSB were rejoined within one hour. During excision repair of DNA damage from u.v.-radiation, the frequencies of DNA SSB as estimated by the alkaline elution technique, were similar in all cell types. Repair replication as measured by BND cellulose chromatography was also similar in epithelial and fibroblastic cells after u.v.-irradiation. Similar levels of SSB were also observed in epithelial and fibroblastic cells after exposure to chemical carcinogens: 7,12-dimethylbenz[a]anthracene; benzo[a]pyrene diol epoxide (BPDE); or N-methyl-N-nitro-N-nitrosoguanidine. Significant repair replication of BPDE-induced DNA damage was detected in both bronchial epithelial and fibroblastic cells, although the level in fibroblasts was approximately 40% of that in epithelial cells. The pulmonary carcinogen asbestos did not damage DNA. DNA-protein crosslinks induced by formaldehyde were rapidly removed in bronchial cells. Further, epithelial and fibroblastic cells, which were incubated with formaldehyde and the polymerase inhibitor combination of cytosine arabinoside and hydroxyurea, accumulated DNA SSB at approximately equal frequencies. These results should provide a useful background for further investigations of the response of human bronchial cells to various DNA damaging agents

  12. DNA-Protected Silver Clusters for Nanophotonics

    Directory of Open Access Journals (Sweden)

    Elisabeth Gwinn

    2015-02-01

    Full Text Available DNA-protected silver clusters (AgN-DNA possess unique fluorescence properties that depend on the specific DNA template that stabilizes the cluster. They exhibit peak emission wavelengths that range across the visible and near-IR spectrum. This wide color palette, combined with low toxicity, high fluorescence quantum yields of some clusters, low synthesis costs, small cluster sizes and compatibility with DNA are enabling many applications that employ AgN-DNA. Here we review what is known about the underlying composition and structure of AgN-DNA, and how these relate to the optical properties of these fascinating, hybrid biomolecule-metal cluster nanomaterials. We place AgN-DNA in the general context of ligand-stabilized metal clusters and compare their properties to those of other noble metal clusters stabilized by small molecule ligands. The methods used to isolate pure AgN-DNA for analysis of composition and for studies of solution and single-emitter optical properties are discussed. We give a brief overview of structurally sensitive chiroptical studies, both theoretical and experimental, and review experiments on bringing silver clusters of distinct size and color into nanoscale DNA assemblies. Progress towards using DNA scaffolds to assemble multi-cluster arrays is also reviewed.

  13. DNA encoding a DNA repair protein

    Science.gov (United States)

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-08-15

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  14. Tomato protoplast DNA transformation : physical linkage and recombination of exogenous DNA sequences

    NARCIS (Netherlands)

    Jongsma, Maarten; Koornneef, Maarten; Zabel, Pim; Hille, Jacques

    1987-01-01

    Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There w

  15. Electrochemical DNA sandwich assay with a lipase label for attomole detection of DNA

    DEFF Research Database (Denmark)

    Ferapontova, Elena; Hansen, Majken Nørgaard; Saunders, Aaron Marc;

    2010-01-01

    A fast and sensitive electrochemical lipase-based sandwich hybridization assay for detection of attomole levels of DNA has been developed. A combination of magnetic beads, used for pre-concentration and bioseparation of the analyte with a lipase catalyst label allowed detection of DNA with a limit...

  16. Tight-binding parameters for charge transfer along DNA

    OpenAIRE

    Hawke, L. G.D.; Kalosakas, G.; Simserides, C.

    2009-01-01

    We systematically examine all the tight-binding parameters pertinent to charge transfer along DNA. The $\\pi$ molecular structure of the four DNA bases (adenine, thymine, cytosine, and guanine) is investigated by using the linear combination of atomic orbitals method with a recently introduced parametrization. The HOMO and LUMO wavefunctions and energies of DNA bases are discussed and then used for calculating the corresponding wavefunctions of the two B-DNA base-pairs (adenine-thymine and gua...

  17. 天门地区152例系统性红斑狼疮患者血清ANA、抗dsDNA抗体和抗ENA抗体联合检测分析%Analysis of combined detection of ANA, anti dsDNA antibody and anti ENA antibody in 152 patients with systemic lupus erythematosus serum in Tianmen area

    Institute of Scientific and Technical Information of China (English)

    郭飞波

    2016-01-01

    ObjectiveTo evaluate the diagnostic valve of combined detection of ANA, anti-dsDNA antibody and anti-ENA antibody in systemic lupus enythmatosus (SLE).MethodFrom April 2012 to April 2015, 152 SLE patients (SLE group) and 85 healthy people (control group) in our hospital were examined and compared. ANA was tested by indirect immumolfuorescence method; anti dsDNA antibody was tested by dot-immunogold filtration assay; anti-ENA antibody was tested by immunoblotting.ResultThe positive rate of combined detection in SLE group was signiifcantly higher than control group (P<0.01). The positive rates of ANA, anti-dsDNA antibody and anti-ENA antibody in SLE group were higher than control group (P<0.05). Among anti-ENA antibody repertoire, the positive rate of anti-SS-A antibody was the highest. The anti-dsDNA positive rate of LN group was higher than NLN group (P<0.05). The proportion of women with autoantibodies was signiifcantly higher than that of men (P<0.01), and the highest positive rate was 56 to 65 years old.Conclusion Combined detection of ANA, anti-dsDNA and anti-ENA antibody has some clinical value of early diagnosis of SLE. Combined testing will help improve the detection rate, and there are signiifcant differences in positive rates of different gender and age of the patients.%目的:探讨联合检测血清抗核抗体(ANA)、抗双链DNA(dsDNA)抗体和抗可提取性核抗原(ENA)抗体对系统性红斑狼疮(SLE)的诊断价值。方法分别采用间接免疫荧光法、胶体金斑点渗滤法、免疫印迹法检测并分析本院2012年4月至2015年4月收治的152例SLE患者(SLE组)与同期85例健康者(对照组)的ANA、抗dsDNA抗体、抗ENA抗体。结果 SLE组患者三种抗体联合检测的阳性率明显高于对照组(P<0.01),且ANA、抗dsDNA抗体、抗ENA抗体的阳性率均明显高于对照组(P<0.05)。在抗ENA抗体谱中,抗SS-A抗体的阳性率最高。狼疮性肾炎(LN)组的

  18. Mechanism of DNA loading by the DNA repair helicase XPD.

    Science.gov (United States)

    Constantinescu-Aruxandei, Diana; Petrovic-Stojanovska, Biljana; Penedo, J Carlos; White, Malcolm F; Naismith, James H

    2016-04-01

    The xeroderma pigmentosum group D (XPD) helicase is a component of the transcription factor IIH complex in eukaryotes and plays an essential role in DNA repair in the nucleotide excision repair pathway. XPD is a 5' to 3' helicase with an essential iron-sulfur cluster. Structural and biochemical studies of the monomeric archaeal XPD homologues have aided a mechanistic understanding of this important class of helicase, but several important questions remain open. In particular, the mechanism for DNA loading, which is assumed to require large protein conformational change, is not fully understood. Here, DNA binding by the archaeal XPD helicase fromThermoplasma acidophilumhas been investigated using a combination of crystallography, cross-linking, modified substrates and biochemical assays. The data are consistent with an initial tight binding of ssDNA to helicase domain 2, followed by transient opening of the interface between the Arch and 4FeS domains, allowing access to a second binding site on helicase domain 1 that directs DNA through the pore. A crystal structure of XPD fromSulfolobus acidocaldiariusthat lacks helicase domain 2 has an otherwise unperturbed structure, emphasizing the stability of the interface between the Arch and 4FeS domains in XPD. PMID:26896802

  19. The improvement of liposome-mediated transfection of pEGFP DNA into human prostate cancer cells by combining low-frequency and low-energy ultrasound with microbubbles.

    Science.gov (United States)

    Bai, Wen-Kun; Wu, Zuo-Hui; Shen, E; Zhang, Ji-Zhen; Hu, Bing

    2012-02-01

    The aim of this study was to explore the use of a contrast agent to study the effects of exposure to ultrasound, in combination with microbubbles, on liposome-mediated transfection of genes into human prostate cancer cells. A contrast agent was used to study the effects of ultrasound exposure in combination with microbubbles on liposomes, which transfect genes into human prostate cancer cells. The human prostate cancer cell line PC-3 in suspension was exposed to ultrasound with a 20% duty cycle (i.e., 2 sec 'on' time and 8 sec 'off' time) lasting 5 min, with and without ultrasound contrast agent (SonoVue™) using a digital sonifier at a frequency of 21 kHz and an intensity of 4.6 mW/cm2. Immediately after exposure to ultrasound, cell viability and membrane damage were measured. After exposure to ultrasound, the cell suspensions were put into 12‑well plates and cultured for 24 h. Fluorescence microscopy and flow cytometry were used to detect pEGFP transfection efficiency. Exposure to ultrasound alone and ultrasound combined with microbubbles resulted in minimal cell death and induced negligible cell membrane damage. Ultrasound combined with microbubbles had a greater effect on cell membrane damage in all groups: the average cell membrane damage was 41.87%, and it was approximately 42‑fold greater than in the control group. The average transfection efficiency of PC-3 cells was 20.30% for the liposome (Lipofectamine™)+pEGFP+ultrasound+ultrasound contrast agent (SonoVue) group; this was the highest rate of all groups measured and was approximately 81‑fold greater than that of the control group. The use of low-frequency and low-energy ultrasound, in combination with microbubbles, could be a potent physical method for increasing liposome gene delivery efficiency. This technique is a promising non-viral approach that can be used in prostate cancer gene therapy.

  20. Combined Effects of Hyperhomocysteinemia and Hypercholesterolemia on Genomic DNA Methylation in Wistar Rats%高同型半胱氨酸和高胆固醇血症联合作用对大鼠基因组DNA总甲基化状态的影响

    Institute of Scientific and Technical Information of China (English)

    王义勇; 罗彩琴; 丛广志; 贾绍斌

    2012-01-01

    目的:探讨高同型半胱氨酸血症(HHCY)和高胆固醇血症(HTC)联合作用对大鼠主动脉基因组DNA总甲基转移酶活力(DNMT)及总甲基化水平的影响.方法:44只健康清洁级成年Wistar雄性大鼠按2×2析因设计随机分为阴性对照组、高同型半胱氨酸(HCY)组、高总胆固醇(TC)组、高HCY+高TC组,每组11只.对照组给予普通大鼠饲料,其余各组给予相应的配方饲料.喂养3个月后心脏取血检测血清中HCY、TC等相关指标.提取主动脉基因组DNA检测基因组DNA总甲基化水平;提取主动脉核蛋白检测基因组DNA总甲基转移酶活力.结果:HHCY和HTC联合作用大鼠血清HCY较高,产生相可作用(P<0.01),表现为协同作用,而对大鼠血清TC、三酰甘油(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)无交互作用.HHCY和HTC联合作用主动脉基因组DNA总甲基转移酶活力较高,产生相互作用(P<0.01),表现为协同作用,总基因组DNA甲基化水平降低,即去甲基化程度的增加,产生相互作用(P<0.01),表现为协同作用.结论:HHCY和HTC联合作用使得大鼠主动脉基因组DNA总DNMT活力增高,基因组DNA总甲基化水平降低,可能是AS发生发展的重要机制之一.%Objective: To study the combined effects of hyperhomocysteinemia (HHCY) and hypercholesterolemia(HTC) on genomic DNA methylation and DNA methyltransferase (DNMT) activity in aortic tissue of Wistar rats. Methods: Forty-four healthy clean level male Wistar rats were randomized into four groups in a 2×2 factorial design, negative control group, high-homocysteine (HCY) group,high-total cholesterol (TC) group and high HCY+TC group. The rats were fed a normal chawdiet in negative control group, and rats of other three groups were fed the formula diet. The serum levels of HCY and TC were detected in rats 3 months after feeding. The aortic genomic DNA was extracted for detection of genomic DNA methylation. The aortic

  1. EFFECT OF CYTOMEGALOVIRUS INFECTION ON THE PREDICTIVE VALUE OF CD-40 LIGAND IN HEART TRANSPLANT RECIPIENTS

    Directory of Open Access Journals (Sweden)

    O. P. Shevchenko

    2012-01-01

    Full Text Available Cytomegalovirus infection may be associated with the development of acute cellular rejection – the most fre- quent and serious complication after heart transplantation, limiting long-term survival of recipients. Biomarkers of inflammation and thrombosis, one of which is the product of platelet activation – a soluble CD40 ligand (sCD40L, also play an important role in the immunopathology of acute rejection. The aim of the study was to assess the risk of cardiovascular complications after heart transplantation under the combined effect of two factors – sCD40L and cytomegalovirus infection. We examined 64 heart recipients in the period of 12 years after heart transplantation. It was revealed that with the presence of elevated levels of sCD40L in combination with cytomegalovirus infection, risk of acute cellular rejection is higher. In recipients with low levels of sCD40L and without cytomegalovirus infection survival rate is significantly higher than in recipients with the presence of one or both of the studied risk factors. 

  2. DNA loops and semicatenated DNA junctions

    Directory of Open Access Journals (Sweden)

    Strauss François

    2000-07-01

    Full Text Available Abstract Background Alternative DNA conformations are of particular interest as potential signals to mark important sites on the genome. The structural variability of CA microsatellites is particularly pronounced; these are repetitive poly(CA · poly(TG DNA sequences spread in all eukaryotic genomes as tracts of up to 60 base pairs long. Many in vitro studies have shown that the structure of poly(CA · poly(TG can vary markedly from the classical right handed DNA double helix and adopt diverse alternative conformations. Here we have studied the mechanism of formation and the structure of an alternative DNA structure, named Form X, which was observed previously by polyacrylamide gel electrophoresis of DNA fragments containing a tract of the CA microsatellite poly(CA · poly(TG but had not yet been characterized. Results Formation of Form X was found to occur upon reassociation of the strands of a DNA fragment containing a tract of poly(CA · poly(TG, in a process strongly stimulated by the nuclear proteins HMG1 and HMG2. By inserting Form X into DNA minicircles, we show that the DNA strands do not run fully side by side but instead form a DNA knot. When present in a closed DNA molecule, Form X becomes resistant to heating to 100°C and to alkaline pH. Conclusions Our data strongly support a model of Form X consisting in a DNA loop at the base of which the two DNA duplexes cross, with one of the strands of one duplex passing between the strands of the other duplex, and reciprocally, to form a semicatenated DNA junction also called a DNA hemicatenane.

  3. Combination Light

    Science.gov (United States)

    1990-01-01

    The Rayovac TANDEM is an advanced technology combination work light and general purpose flashlight that incorporates several NASA technologies. The TANDEM functions as two lights in one. It features a long range spotlight and wide angle floodlight; simple one-hand electrical switching changes the beam from spot to flood. TANDEM developers made particular use of NASA's extensive research in ergonomics in the TANDEM's angled handle, convenient shape and different orientations. The shatterproof, water resistant plastic casing also draws on NASA technology, as does the shape and beam distance of the square diffused flood. TANDEM's heavy duty magnet that permits the light to be affixed to any metal object borrows from NASA research on rare earth magnets that combine strong magnetic capability with low cost. Developers used a NASA-developed ultrasonic welding technique in the light's interior.

  4. Functionalizing Designer DNA Crystals

    Science.gov (United States)

    Chandrasekaran, Arun Richard

    Three-dimensional crystals have been self-assembled from a DNA tensegrity triangle via sticky end interaction. The tensegrity triangle is a rigid DNA motif containing three double helical edges connected pair-wise by three four-arm junctions. The symmetric triangle contains 3 unique strands combined in a 3:3:1 ratio: 3 crossover, 3 helical and 1 central. The length of the sticky end reported previously was two nucleotides (nt) (GA:TC) and the motif with 2-helical turns of DNA per edge diffracted to 4.9 A at beam line NSLS-X25 and to 4 A at beam line ID19 at APS. The purpose of these self-assembled DNA crystals is that they can be used as a framework for hosting external guests for use in crystallographic structure solving or the periodic positioning of molecules for nanoelectronics. This thesis describes strategies to improve the resolution and to incorporate guests into the 3D lattice. The first chapter describes the effect of varying sticky end lengths and the influence of 5'-phosphate addition on crystal formation and resolution. X-ray diffraction data from beam line NSLS-X25 revealed that the crystal resolution for 1-nt (G:C) sticky end was 3.4 A. Motifs with every possible combination of 1-nt and 2-nt sticky-ended phosphorylated strands were crystallized and X-ray data were collected. The position of the 5'-phosphate on either the crossover (strand 1), helical (strand 2), or central strand (3) had an impact on the resolution of the self-assembled crystals with the 1-nt 1P-2-3 system diffracting to 2.62 A at APS and 3.1 A at NSLS-X25. The second chapter describes the sequence-specific recognition of DNA motifs with triplex-forming oligonucleotides (TFOs). This study examined the feasibility of using TFOs to bind to specific locations within a 3-turn DNA tensegrity triangle motif. The TFO 5'-TTCTTTCTTCTCT was used to target the tensegrity motif containing an appropriately embedded oligopurine.oligopyrimidine binding site. As triplex formation involving cytidine

  5. DNA定量分析联合阴道镜在诊断宫颈癌及高级别宫颈上皮内瘤变中的价值%Value of DNA quantitative analysis combined with colposcopy for diagnosis of cervical cancer and high- grade cervical intraepithelial neoplasia

    Institute of Scientific and Technical Information of China (English)

    李苗; 刘芳芳; 邸媛媛; 邓清华; 钟培根

    2012-01-01

    目的:探讨宫颈细胞DNA定量分析法联合阴道镜在宫颈癌及高级别宫颈上皮内瘤变(CIN)诊断中的价值.方法:对4 577名妇女的宫颈细胞采用DNA定量分析系统和常规细胞学进行检查,常规细胞学诊断低级别鳞状上皮内瘤变(LSIL)及以上级别或DNA定量分析异常者在阴道镜下行宫颈活组织病理检查.结果:50名妇女做了病理活检,病理诊断宫颈癌及CIN 2以上病变共27例,其中DNA定量分析方法异常25例(92.6%),常规细胞学异常6例(22.2%),阴道镜异常22例(81.5%),三者间差异有统计学意义(P<0.005).DNA定量分析及阴道镜检查筛查子宫颈癌及高级别宫颈上皮内瘤变(≥CIN2)的敏感度、特异度均高于常规细胞学.结论:DNA定量分析方法联合阴道镜在早期诊断宫颈癌及高级别宫颈上皮内瘤变方面有更高的敏感性,适合在基层单位大力推广.%Objective: To explore the value of DNA quantitative analysis of cervical cells combined with colposcopy for diagnosis of cervical cancer and high-grade cervical intraepithelial neoplasia (CIN) . Methods: The cervical cells of 4 577 women were examined by DNA quantitative analysis system and routine cytologic test, the women diagnosed as low - grade squamous intraepithelial lesion (LSIL) and lesions above by routine cytologic test and the women with abnormal results of DNA quantitative analysis underwent cervical biopsy under col-poscope and pathological examination. Results: Fifty women underwent pathological biopsy, and 27 women were diagnosed as cervical cancer and &CIN Ⅱ lesions, 25 women (92. 6% ) were found with abnormal results of DNA quantitative analysis, 6 women (22. 2% ) were found with abnormal results of routine cytologic test, 22 women (81. 5% ) were found with abnormal results of colposcopy, there was statistically significant difference among the three rates (P < 0. 005 ) . The sensitivities and specificities of DNA quantitative analysis and

  6. 胎儿颈项透明层联合中孕产前无创DNA检测在胎儿染色体疾病产前筛查中的应用%Application of nuchal translu-cency in combination with non-invasive prenatal DNA test in fetal chromosomal disease

    Institute of Scientific and Technical Information of China (English)

    刘灿; 王坤; 李丽霞; 王明辉; 徐翠; 张风琴; 卢克新

    2015-01-01

    目的:探讨胎儿颈项透明层联合产前无创DNA检测在胎儿染色体疾病产前筛查中的临床应用价值.方法:2010年10月至2014年6月我院妇产科门诊就诊的孕11 ~ 14周孕妇713例,测量胎儿颈项透明层厚度,厚度异常的孕妇根据自愿行无创DNA检测,并随访观察. 结果: 713例孕妇超声检查提示胎儿颈项透明层增厚27例,21例行无创DNA检查,21-三体3例,18-三体1例,17例染色体核型正常的胎儿在以后的超声检查随访中6例出现异常. 6例未行无创DNA检查的孕妇在以后的超声检查随访中2例出现异常;21-三体胎儿的颈项透明层厚度范围为3.7 ~ 4.4 mm, 平均4.0 mm;1例18-三体胎儿的颈项透明层厚度约5.0 mm.结论:胎儿颈项透明层厚度联合无创DNA 检测能够提高胎儿染色体疾病的检出率,减少染色体异常胎儿出生率.%Objective To assess the value of nuchal translu-cency in combination with non-invasive prenatal DNA test in fetal with chromosomal disease. Methods A total of 713 cases with single pregnancies, with 11 ~ 14 gestational weeks, were enrolled in this study. We measured the thickness of nuchal translu-cency, If the thickness was abnomal, the pregnant women would receive the non-invasive prenatal DNA test on voluntary basis and were followed up. Results There were 27 cases with abnormal NT among 713 cases. Twenty-one cases received non-invasive prenatal DNA test , the test results showed that 3 cases were trisomy 21 , 1 cases was trisomy 18. Among the 17 cases with normal chromosome karyotype, 6 cases were found abnomal during the follw-up. Two cases were found abnomal among the 6 cases undergo the non-invasive prenatal DNA test.The thickness of NT, ranging from 3.7 mm to 4.4 mm, was trisomy 21, with the average of 4.0 mm, and the thickness of NT was 5.0mm and was trisomy 18. Conclusions The application of nuchal translu-cency in combination with non-invasive prenatal DNA test could improve the ability to find

  7. Salt-Dependent DNA-DNA Spacings in Intact Bacteriophage lambda Reflect Relative Importance of DNA Self-Repulsion and Bending Energies

    Energy Technology Data Exchange (ETDEWEB)

    X Qiu; D Rau; V Parsegian; L Fang; C Knobler; W Gelbart

    2011-12-31

    Using solution synchrotron x-ray scattering, we measure the variation of DNA-DNA d spacings in bacteriophage {lambda} with mono-, di-, and polyvalent salt concentrations, for wild-type [48.5 x 10{sup 3} base pairs (bp)] and short-genome-mutant (37.8 kbp) strains. From the decrease in d spacings with increasing salt, we deduce the relative contributions of DNA self-repulsion and bending to the energetics of packaged phage genomes. We quantify the DNA-DNA interaction energies within the intact phage by combining the measured d spacings in the capsid with measurements of osmotic pressure in DNA assemblies under the same salt conditions in bulk solution. In the commonly used Tris-Mg buffer, the DNA-DNA interaction energies inside the phage capsids are shown to be about 1 kT/bp, an order of magnitude larger than the bending energies.

  8. Eupolybothrus cavernicolus Komerički & Stoev sp. n. (Chilopoda: Lithobiomorpha: Lithobiidae: the first eukaryotic species description combining transcriptomic, DNA barcoding and micro-CT imaging data

    Directory of Open Access Journals (Sweden)

    Pavel Stoev

    2013-10-01

    Full Text Available We demonstrate how a classical taxonomic description of a new species can be enhanced by applying new generation molecular methods, and novel computing and imaging technologies. A cave-dwelling centipede, Eupolybothrus cavernicolus Komerički & Stoev sp. n. (Chilopoda: Lithobiomorpha: Lithobiidae, found in a remote karst region in Knin, Croatia, is the first eukaryotic species for which, in addition to the traditional morphological description, we provide a fully sequenced transcriptome, a DNA barcode, detailed anatomical X-ray microtomography (micro-CT scans, and a movie of the living specimen to document important traits of its ex-situ behaviour. By employing micro-CT scanning in a new species for the first time, we create a high-resolution morphological and anatomical dataset that allows virtual reconstructions of the specimen and subsequent interactive manipulation to test the recently introduced ‘cybertype’ notion. In addition, the transcriptome was recorded with a total of 67,785 scaffolds, having an average length of 812 bp and N50 of 1,448 bp (see GigaDB. Subsequent annotation of 22,866 scaffolds was conducted by tracing homologs against current available databases, including Nr, SwissProt and COG. This pilot project illustrates a workflow of producing, storing, publishing and disseminating large data sets associated with a description of a new taxon. All data have been deposited in publicly accessible repositories, such as GigaScience GigaDB, NCBI, BOLD, Morphbank and Morphosource, and the respective open licenses used ensure their accessibility and re-usability.

  9. ANA联合dsDNA在自身免疫疾病中的相关性和敏感性分析%Analysis on the Correlation and Sensitivity of ANA combined with dsDNA in Autoimmune Diseases

    Institute of Scientific and Technical Information of China (English)

    陈英杰; 曹令仪; 雷泽洪; 冯武超; 钟玉琼

    2015-01-01

    目的 探讨ANA联合dsDNA在自身免疫疾病中的相关性和敏感性.方法 选择2014年6月至2014年12月我院收治的自身免疫性疾病患者466例.采用间接免疫荧光法检测ANA、ELISA法检测dsDNA.观察ANA阳性患者ANA核型,SLE、MCTD等疾病患者ANA、dsDNA阳性率,以及不同疾病程度SLE患者血清ANA、dsDNA计分值.结果 184例ANA核型阳性患者中以核均质型构成比例最高,占33.15%,其次为胞浆型、核细颗粒型和核粗颗粒型,分别占20.11%、19.02%和16.30%.ANA阳性率从高到低分别是SLE、MCTD、PSS;dsDNA阳性率从高到低分别是SLE、MCTD.非活动期SLE患者ANA、dsDNA计分值明显低于中度活期组和重度活动期组,中度活动期组患者dsDNA计分值明显低于重度活动期组,差异具有统计学意义(P<0.05).结论 ANA是诊断自身免疫疾病高度敏感的抗体,联合dsDNA可提高鉴别诊断准确性,为临床提供有价值的参考.

  10. Enzyme-guided DNA Sewing Architecture.

    Science.gov (United States)

    Song, In Hyun; Shin, Seung Won; Park, Kyung Soo; Lansac, Yves; Jang, Yun Hee; Um, Soong Ho

    2015-12-04

    With the advent of nanotechnology, a variety of nanoarchitectures with varied physicochemical properties have been designed. Owing to the unique characteristics, DNAs have been used as a functional building block for novel nanoarchitecture. In particular, a self-assembly of long DNA molecules via a piece DNA staple has been utilized to attain such constructs. However, it needs many talented prerequisites (e.g., complicated computer program) with fewer yields of products. In addition, it has many limitations to overcome: for instance, (i) thermal instability under moderate environments and (ii) restraint in size caused by the restricted length of scaffold strands. Alternatively, the enzymatic sewing linkage of short DNA blocks is simply designed into long DNA assemblies but it is more error-prone due to the undeveloped sequence data. Here, we present, for the first time, a comprehensive study for directly combining DNA structures into higher DNA sewing constructs through the 5'-end cohesive ligation of T4 enzyme. Inspired by these achievements, the synthesized DNA nanomaterials were also utilized for effective detection and real-time diagnosis of cancer-specific and cytosolic RNA markers. This generalized protocol for generic DNA sewing is expected to be useful in several DNA nanotechnology as well as any nucleic acid-related fields.

  11. DNA extraction by zinc.

    OpenAIRE

    Kejnovský, E; Kypr, J

    1997-01-01

    A fast, very simple and efficient method of DNA extraction is described which takes advantage of DNA sedimentation induced by millimolar concentrations of ZnCl2. The zinc-induced sedimentation is furthermore strongly promoted by submillimolar phosphate anion concentrations. Within 90% of DNA irrespective of whether a plasmid DNA or short oligonucleotides are the extracted material. The method works with plasmid DNA and oligonucleotide concentrations as low as 100 ng/ml and 10 microg/ml, respe...

  12. Purification of total DNA extracted from activated sludge

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Purification of the total DNA extracted from activated sludge samples was studied. The effects of extraction buffers and lysis treatments (lysozyme, sodium dodecyl sulfate (SDS), sonication, mechanical mill and thermal shock) on yield and purity of the total DNA extracted from activated sludge were investigated. It was found that SDS and mechanical mill were the most effective ways for cell lysis, and both gave the highest DNA yields, while by SDS and thermal shock, the purest DNA extract could be obtained. The combination of SDS with other lysis treatment, such as sonication and thermal shock, could apparently increase the DNA yields but also result in severe shearing. For the purification of the crude DNA extract, polyvinyl polypyrrolidone was used for the removal of humic contaminants. Cetyltrimethyl ammonium bromide, potassium acetate and phenol/chloroform were used to remove proteins and polysaccharides from crude DNA. Crude DNA was further purified by isopropanol precipitation. Thus, a suitable protocol was proposed for DNA extraction, yielding about 49.9 mg (DNA)/g volatile suspended solids, and the DNA extracts were successfully used in PCR amplifications for 16S rDNA and 16S rDNA V3 region. The PCR products of 16S rDNA V3 region allowed the DGGE analysis (denatured gradient gel electrophoresis) to be possible.

  13. Mechanical force-induced DNA damage during AFM single-molecule manipulation

    International Nuclear Information System (INIS)

    Many environmental factors can cause DNA damage, such as radiation, heat, oxygen free radical, etc., which can induce mutation during DNA replication. Meanwhile, DNA molecules are subjected to various mechanical forces in numerous biological processes. However, it is unknown whether the mechanical force would induce DNA damage and introduce mutation during DNA replication, With the combination of single-molecule manipulation based on atomic force microscopy (AFM), single molecular polymerase chain reaction (SM-PCR) and Sanger's sequencing, we investigated the effect of mechanical force on DNA. The results show that mechanical force can cause DNA damage and induce DNA mutation during amplification. (authors)

  14. DNA polymerase δ and DNA repair: DNA repair synthesis in human fibroblasts requires DNA polymerase δ

    International Nuclear Information System (INIS)

    When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernate of similarly treated HeLa cells. Monoclonal antibody to KB cell DNA polymerase α, while binding to HeLa DNA polymerase α, did not bind to the HeLa DNA polymerase δ. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGT) and 2(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase α, but did not inhibit the DNA polymerase δ. Neither purified DNA polymerase α nor β could promote repair DNA synthesis in the permeabilized cells. Furthermore, if monoclonal antibodies to DNA polymerase α BuPdGTP, or BuAdATP was added to the reconstituted system, there was no significant inhibition

  15. Combined Detection of Hepatitis B five,Pre S1 antigen and HBV-DNA Value in Clinical Application%联合检测乙型病毒性肝炎五项、前S1抗原与HBV-DNA的临床应用价值

    Institute of Scientific and Technical Information of China (English)

    刘绮婷; 黎阳成; 何秋贤

    2015-01-01

    目的 探讨联合检测乙型病毒性肝炎五项、前S1抗原与乙型肝炎病毒脱氧核糖核酸(HBV-DNA)的临床关系,评估其在乙型肝炎病毒(HBV)检测中的临床价值.方法 将2011年9月至2013年9月于我院接受治疗的100例乙型病毒性肝炎表面抗原阳性患者作为研究对象,收集其临床资料,采集所有患者肘静脉血液,并选用定量法与酶联免疫吸附试验进行乙型病毒性肝炎五项、前S1抗原及HBV-DNA检测,评估其临床价值.结果 在不同的乙型病毒性肝炎五项模式下,前S1抗原与HBV-DNA检测结果比较差异无统计学意义(P>0.05);两组患者HBV-DNA与乙型病毒性肝炎前S1抗原检出符合率比较差异无统计学意义(P>0.05).结论 选用乙型病毒性肝炎前S1抗原与HBV-DNA联合检测能提高乙型病毒性肝炎的检出率,指导其治疗与预后有重要意义.%Objective To investigate the combined detection of hepatitis B five,pre-S1 antigen clinical relationship with HBV-DNA to assess the clinical value of hepatitis B virus(HBV)detection.Methods The September 2011 to September 2013 in our hospital treated 100 cases of hepatitis B surface antigen-positive patients as research subjects,the colection of clinical data colected for al patients cubital vein blood,and the choice of method and quantitative enzyme-linked immunosorbent assay the hepatitis B five,pre-S1 antigen and HBV-DNA detection method to assess the clinical relationship.Results Hepatitis B in five different models,pre-S1 antigen and HBV-DNA test results showed no significant difference(P>0.05);patients were HBV-DNA and HBV pre-S1 antigen detection rate is relatively consistent with the difference was not statisticaly significant(P>0.05).Conclusion Pre-selection of hepatitis B antigen and HBV-DNA S1 joint detection of hepatitis B to improve the detection rate,to guide their treatment and prognosis are important.

  16. 腹腔积液细胞DNA倍体及miRNA-21联合检测对肝细胞癌诊断的临床价值%Diagnostic value of combined determination of DNA ploidy level and miRNA-2 1 expression in ascites cells for hepa-tocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    肖作汉; 孟冈; 王立志; 孙文锦

    2015-01-01

    目的:评估联合检测腹腔积液细胞DNA倍体与微小RNA-21(miRNA-21)表达诊断肝细胞癌(HCC)的效能。方法将2012年1月1日-2014年1月1日鄂州市中心医院所收治临床表现伴有腹腔积液的患者纳入研究,HCC患者分入试验组,非HCC患者分入对照组。比较两组患者相关危险因素指标的差异性,采用Logistic回归分析进一步提取独立敏感指标,绘制相应指标的ROC曲线,计算曲线下面积(AUC)及约登指数(YI)。结果两组患者组间资料单因素比较显示:性别(男性比例)、吸烟史、HBV阳性、上腹部CT疑似阳性、卡氏功能量表(KPS量表)、ALT、甲胎蛋白(AFP)、异常凝血酶原(APT)、α-L-岩藻糖苷酶(AFU )、腹水细胞miRNA-21、腹水细胞DNA异倍体阳性共计11项指标差异存在统计学意义(P值均<0.05)。Logistic回归分析显示,吸烟史、AFP、APT、腹水细胞miRNA-21、腹水细胞DNA异倍体是诊断HCC的敏感指标。腹水细胞miRNA-21与DNA异倍体的AUC分别为0.742与0.801;吸烟史、AFP与APT三者联合断HCC的AUC为0.852,YI为0.5992。吸烟史、AFP、APT腹水细胞miRNA-21与DNA异倍体五项指标联合诊断的AUC增至0.869、YI增至0.6222。结论腹水细胞DNA异倍体与miRNA-21是诊断HCC的独立危险因素,单项指标检测即具有良好的诊断价值,将其联用能够显著增大HCC的诊断价值,对于该病的早期诊断具有重要临床意义,是当前HCC诊断体系的良好补充。%Objective To evaluate the diagnostic value of combined determination of DNA ploidy level and microRNA-21 (miRNA-21) expression in ascites cells for hepatocellular carcinoma (HCC).Methods A total of 1 17 patients with ascites admitted to our hospital from January 1,2012 to January 1,2014 were enrolled.The patients with a confirmed diagnosis of HCC were assigned to experimental group (n=48),and those without

  17. Functionalizing Designer DNA Crystals

    Science.gov (United States)

    Chandrasekaran, Arun Richard

    Three-dimensional crystals have been self-assembled from a DNA tensegrity triangle via sticky end interaction. The tensegrity triangle is a rigid DNA motif containing three double helical edges connected pair-wise by three four-arm junctions. The symmetric triangle contains 3 unique strands combined in a 3:3:1 ratio: 3 crossover, 3 helical and 1 central. The length of the sticky end reported previously was two nucleotides (nt) (GA:TC) and the motif with 2-helical turns of DNA per edge diffracted to 4.9 A at beam line NSLS-X25 and to 4 A at beam line ID19 at APS. The purpose of these self-assembled DNA crystals is that they can be used as a framework for hosting external guests for use in crystallographic structure solving or the periodic positioning of molecules for nanoelectronics. This thesis describes strategies to improve the resolution and to incorporate guests into the 3D lattice. The first chapter describes the effect of varying sticky end lengths and the influence of 5'-phosphate addition on crystal formation and resolution. X-ray diffraction data from beam line NSLS-X25 revealed that the crystal resolution for 1-nt (G:C) sticky end was 3.4 A. Motifs with every possible combination of 1-nt and 2-nt sticky-ended phosphorylated strands were crystallized and X-ray data were collected. The position of the 5'-phosphate on either the crossover (strand 1), helical (strand 2), or central strand (3) had an impact on the resolution of the self-assembled crystals with the 1-nt 1P-2-3 system diffracting to 2.62 A at APS and 3.1 A at NSLS-X25. The second chapter describes the sequence-specific recognition of DNA motifs with triplex-forming oligonucleotides (TFOs). This study examined the feasibility of using TFOs to bind to specific locations within a 3-turn DNA tensegrity triangle motif. The TFO 5'-TTCTTTCTTCTCT was used to target the tensegrity motif containing an appropriately embedded oligopurine.oligopyrimidine binding site. As triplex formation involving cytidine

  18. DNA fragmentation in apoptosis

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Cleavage of chromosomal DNA into oligonucleosomal size fragments is an integral part of apoptosis. Elegant biochemical work identified the DNA fragmentation factor (DFF) as a major apoptotic endonuclease for DNA fragmentation in vitro. Genetic studies in mice support the importance of DFF in DNA fragmentation and possibly in apoptosis in vivo. Recent work also suggests the existence of additional endonucleases for DNA degradation. Understanding the roles of individual endonucleases in apoptosis, and how they might coordinate to degrade DNA in different tissues during normal development and homeostasis, as well as in various diseased states, will be a major research focus in the near future.

  19. Karyotyping of Brassica oleracea L.based on rDNA and Cot-1 DNA fluorescence in situ hybridization

    Institute of Scientific and Technical Information of China (English)

    WANG Taixia; WU Chunhong; HUANG Jinyong; WEI Wenhui

    2007-01-01

    To explore an effective and reliable karyotyping method in Brassica crop plants,Cot-1 DNA was isolated from Brassica oleracea genome,labeled as probe with Biotin-Nick Translation Mix kit,in situ hybridized to mitotic spreads,and where specific fluorescent bands showed on each chromosome pair.25S and 5S rDNA were labeled as probes with DIG-Nick Translation Mix kit and Biotin-Nick Translation Mix kit,respectively,in situ hybridized to mitotic preparations,where 25S rDNA could be detected on two chromosome pairs and 5S rDNA on only one.Cot-1 DNA contains rDNA and chromosome sites identity between Cot-1 DNA and 25S rDNA was determined by dual-colour fluorescence in situ hybridization.All these showed that the karyotyping technique based on a combination of rDNA and Cot-1 DNA chromosome landmarks is superior to all but one.A more exact karyotype ofB.oleracea has been analyzed based on a combination of rDNA sites,Cot-1 DNA fluorescent bands,chromosome lengths and arm ratios.

  20. [Uracil-DNA glycosylases].

    Science.gov (United States)

    Pytel, Dariusz; Słupianek, Artur; Ksiazek, Dominika; Skórski, Tomasz; Błasiak, Janusz

    2008-01-01

    Uracil is one of four nitrogen bases, most frequently found in normal RNA. Uracyl can be found also in DNA as a result of enzymatic or non-enzymatic deamination of cytosine as well as misincorporation of dUMP instead of dTMP during DNA replication. Uracil from DNA can be removed by DNA repair enzymes with apirymidine site as an intermediate. However, if uracil is not removed from DNA a pair C:G in parental DNA can be changed into a T:A pair in the daughter DNA molecule. Therefore, uracil in DNA may lead to a mutation. Uracil in DNA, similarly to thymine, forms energetically most favorable hydrogen bonds with adenine, therefore uracil does not change the coding properties of DNA. Uracil in DNA is recognized by uracil DNA glycosylase (UDGs), which initiates DNA base excision repair, leading to removing of uracil from DNA and replacing it by thymine or cytosine, when arose as a result of cytosine deamination. Eukaryotes have at least four nuclear UDGs: UNG2, SMUG1, TDG i MBD4, while UNG1 operates in the mitochondrium. UNG2 is involved in DNA repair associated with DNA replication and interacts with PCNA and RPA proteins. Uracil can also be an intermediate product in the process of antigen-dependent antibody diversification in B lymphocytes. Enzymatic deamination of viral DNA by host cells can be a defense mechanism against viral infection, including HIV-1. UNG2, MBD4 and TDG glycosylases may cooperate with mismatch repair proteins and TDG can be involved in nucleotide excision repair system.

  1. DNA damage and autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely [Redox Biology Center and School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583 (United States); Panayiotidis, Mihalis I. [School of Community Health Sciences, University of Nevada, Reno, NV 89557 (United States); Franco, Rodrigo, E-mail: rfrancocruz2@unl.edu [Redox Biology Center and School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583 (United States)

    2011-06-03

    Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.

  2. DNA Open states and DNA hydratation

    International Nuclear Information System (INIS)

    It is a very well-known fact that an protonic exchange exists among natural DNA filaments and synthetic polynucleotides with the solvent (1--2). The existence of DNA open states, that is to say states for which the interior of the DNA molecule is exposed to the external environment, it has been demonstrated by means of proton-deuterium exchange (3). This work has carried out experiments measuring the dispersion of the traverse relaxation rate (4), as a pulsation rate function in a Carr-Purcell-Meiboom-Gill (CPMG) pulses sequence rate, to determine changes in the moist layer of the DNA molecule. The experiments were carried out under different experimental conditions in order to vary the probability that open states occurs, such as temperature or the exposure to electromagnetic fields. Some theoretical models were supposed to adjust the experimental results including those related to DNA non linear dynamic

  3. Genetic polymorphisms of 9 non-combined of DNA index system short tandem repeat loci in Guangdong Han population%九个非DNA联合索引系统核心短串联重复序列基因座在广东汉族人群的遗传多态性

    Institute of Scientific and Technical Information of China (English)

    张晋湘; 薛天羽; 李海霞; 孙宏钰; 成建定

    2009-01-01

    目的 调查D7S3048等9个非DNA联合索引系统(combined of DNA index system,CODIS)指定的核心短串联重复序列(short tandem repeat,STR)基因座在广东汉族人群的遗传多态性.方法 采用荧光标记复合扩增和毛细管电泳技术,对广东汉族500名无关个体的DNA进行9个STR基因座分型.结果 500名无关个体在D7S3048等9个非CODIS核心STR基因座共检出115个等位基因,160种基因型,各基因座杂合度为0.824~0.884,个人识别能力为0.925~0.969,多态信息总量为0.77~0.86,均符合Hardy-Weinberg平衡(P>0.05),9个STR基因座的累计个体识别力达1.00×10~(-13),三联体累计非父排除率为0.999989488,二联体累计非父排除率为0.873436.结论 D7S3048等9个STR基因座在个体识别及亲子鉴定中是一个高效的检测系统,在二联体亲子鉴定中可作为补充基因座满足疑难、单亲和突变案件的需求.%Objective To investigate the genetic polymorphisms and their forensic application of 9 non-combined of DNA index system (CODIS) short tandem repeat (STR) loci in Guangdong Han population. Methods DNA samples from 500 unrelated individuals were extracted and amplified with fluorescence labeled multiplex PCR system. PCR products were separated and genotyped with capillary electrophoresis. Results One hundred and fifteen alleles and 160 genotypes were observed in the 9 STR loci, respectively. The heterozygosity was 0. 824-0. 884, the discrimination power (DP) was 0. 925-0. 969 and the polymorphism information content (PIC) was 0. 77-0. 86, respectively. The distribution met the Hardy-Weinberg equilibrium (P>0.05). The total discrimination power was 1.00×10~(-13), the combined probability of exclusion for trio-paternity testing was 0. 999989488. The combined probability of exclusion for duo-paternity testing was 0. 873436. Conclusion The 9 STR loci are powerful and reliable for personal identification and paternity testing. They can be used as supplementary loci

  4. Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions

    Science.gov (United States)

    Gardner, Shea N.; Mariella, Jr., Raymond P.; Christian, Allen T.; Young, Jennifer A.; Clague, David S.

    2011-01-18

    A method of fabricating a DNA molecule of user-defined sequence. The method comprises the steps of preselecting a multiplicity of DNA sequence segments that will comprise the DNA molecule of user-defined sequence, separating the DNA sequence segments temporally, and combining the multiplicity of DNA sequence segments with at least one polymerase enzyme wherein the multiplicity of DNA sequence segments join to produce the DNA molecule of user-defined sequence. Sequence segments may be of length n, where n is an even or odd integer. In one embodiment the length of desired hybridizing overlap is specified by the user and the sequences and the protocol for combining them are guided by computational (bioinformatics) predictions. In one embodiment sequence segments are combined from multiple reading frames to span the same region of a sequence, so that multiple desired hybridizations may occur with different overlap lengths. In one embodiment starting sequence fragments are of different lengths, n, n+1, n+2, etc.

  5. Preparation of Phi29 DNA polymerase free of amplifiable DNA using ethidium monoazide, an ultraviolet-free light-emitting diode lamp and trehalose.

    Directory of Open Access Journals (Sweden)

    Hirokazu Takahashi

    Full Text Available We previously reported that multiply-primed rolling circle amplification (MRPCA using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control casts doubt on the result of DNA amplification. Since Phi29 DNA polymerase has high affinity for both single-strand and double-stranded DNA, some amount of host DNA will always remain in the recombinant polymerase. Here we describe a procedure for preparing Phi29 DNA polymerase which is essentially free of amplifiable DNA. This procedure is realized by a combination of host DNA removal using appropriate salt concentrations, inactivation of amplifiable DNA using ethidium monoazide, and irradiation with visible light from a light-emitting diode lamp. Any remaining DNA, which likely exists as oligonucleotides captured by the Phi29 DNA polymerase, is degraded by the 3'-5' exonuclease activity of the polymerase itself in the presence of trehalose, used as an anti-aggregation reagent. Phi29 DNA polymerase purified by this procedure has little amplifiable DNA, resulting in reproducible amplification of at least ten copies of plasmid DNA without any byproducts and reducing reaction volume. This procedure could aid the amplification of tiny amounts DNA, thereby providing clear evidence of contamination from laboratory environments, tools and reagents.

  6. Diagnostic validity of cervical precancerous lesions by liquid-based cytology technique combined with high-risk HPV-DNA detection%液基细胞学技术联合高危HPV-DNA检测对宫颈癌前病变的诊断效度

    Institute of Scientific and Technical Information of China (English)

    王泽曼; 陈玉; 郑建鹏

    2011-01-01

    目的:探讨液基细胞学检查技术(LCT)联合高危人乳头状瘤病毒DNA(HPV-DNA)检测诊断宫颈癌前病变的效度.方法:对2007年12月~2010年12月来我院行体检的19~65岁的6 521名女性采用LCT进行宫颈癌的筛查,以及HPV分型基因芯片检测系统进行18种高危HPV基因亚型检测.对上述检测阳性者行阴道镜下宫颈活组织检查,对检测均阴性者依其意愿进行阴道镜下宫颈活组织检查.结果:LCT阳性(≥ASCUS)152例,HPV阳性86例,其中二者均为阳性的有42例,LCT和HPV均为阴性的有6 325例;LCT阳性的152例和HPV阳性的86例中病理活组织检查结果为阳性(≥CIN I)的分别有112例和68例,其中LCT和HPV均阳性的42例中病理活组织检查阳性的有34例.LCT和HPV均为阴性的6 325例中有2 000人自愿行病理检查,其中1人病理检查结果为阳性.LCT诊断宫颈癌前病变的灵敏度为76.19%,特异度为98.05%;HPV检测诊断宫颈癌前病变的灵敏度为46.26%,特异度为99.12%;两方法联合诊断(其中1项阳性即判定为患者)宫颈癌前病变的灵敏度为99.32%,特异度为99.61%.结论:液基细胞检测技术和高危HPV-DNA检测的联合应用优于单项技术检测,对于宫颈癌前病变的筛检具有重要意义.%AIM: To investigate liquid - based cytology technique ( LCT ) combined with high - risk human papilloma virus DNA ( HPV - DNA ) detection for diagnosis of cervical precancerous lesions. METHODS: Screening of cervical cancer was performed by LCT combined with HPV - DNA detection in 6 521 women at the age of 19 ~65 in our hospital for physical examination from December 2007 to December 2010. Eighteen high - risk HPV isoform genes were detected by HPV typing gene chip detection system. The women with positive detection underwent colposcopic cervical biopsies. The women with negative detection also underwent colposcopic cervical biopsies if the operation was of their own accord. RESULTS: The LCT positive results

  7. Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase

    OpenAIRE

    Manrao, Elizabeth A; Derrington, Ian M.; Laszlo, Andrew H; Langford, Kyle W.; Hopper, Matthew K; Gillgren, Nathaniel; Pavlenok, Mikhail; Niederweis, Michael; Gundlach, Jens H.

    2012-01-01

    Nanopore technologies are being developed for fast and direct sequencing of single DNA molecules through detection of ionic current modulations as DNA passes through a pore’s constriction1,2. Here we demonstrate the ability to resolve changes in current that correspond to a known DNA sequence by combining the high sensitivity of a mutated form of the protein pore Mycobacterium smegmatis porin A (MspA)3 with phi29 DNA polymerase (DNAP)4, which controls the rate of DNA translocation through the...

  8. A DNA extraction protocol for improved DNA yield from individual mosquitoes [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Catelyn C. Nieman

    2015-11-01

    Full Text Available Typical DNA extraction protocols from commercially available kits provide an adequate amount of DNA from a single individual mosquito sufficient for PCR-based assays. However, next-generation sequencing applications and high-throughput SNP genotyping assays exposed the limitation of DNA quantity one usually gets from a single individual mosquito. Whole genome amplification could alleviate the issue but it also creates bias in genome representation. While trying to find alternative DNA extraction protocols for improved DNA yield, we found that a combination of the tissue lysis protocol from Life Technologies and the DNA extraction protocol from Qiagen yielded a higher DNA amount than the protocol using the Qiagen or Life Technologies kit only. We have not rigorously tested all the possible combinations of extraction protocols; we also only tested this on mosquito samples. Therefore, our finding should be noted as a suggestion for improving people’s own DNA extraction protocols and not as an advertisement of a commercially available product.

  9. Modeling DNA Replication.

    Science.gov (United States)

    Bennett, Joan

    1998-01-01

    Recommends the use of a model of DNA made out of Velcro to help students visualize the steps of DNA replication. Includes a materials list, construction directions, and details of the demonstration using the model parts. (DDR)

  10. HPV DNA test

    Science.gov (United States)

    The HPV DNA test is used to check for high-risk HPV infection in women. HPV infection around the genitals is ... warts spread when you have sex. The HPV-DNA test is generally not recommended for detecting low- ...

  11. DNA tagged microparticles

    Science.gov (United States)

    Farquar, George Roy; Leif, Roald N; Wheeler, Elizabeth

    2015-05-05

    A simulant that includes a carrier and DNA encapsulated in the carrier. Also a method of making a simulant including the steps of providing a carrier and encapsulating DNA in the carrier to produce the simulant.

  12. Recombinant DNA in Medicine

    OpenAIRE

    Cederbaum, Stephen D.; Fareed, George C.; Lovett, Michael A.; Shapiro, Larry J.

    1984-01-01

    Studies in bacteria and bacterial viruses have led to methods to manipulate and recombine DNA in unique and reproducible ways and to amplify these recombined molecules millions of times. Once properly identified, the recombinant DNA molecules can be used in various ways useful in medicine and human biology. There are many applications for recombinant DNA technology. Cloned complementary DNA has been used to produce various human proteins in microorganisms. Insulin and growth hormone have been...

  13. DNA Damage Response

    OpenAIRE

    Giglia-Mari, Giuseppina; Zotter, Angelika; Vermeulen, Wim

    2011-01-01

    Structural changes to DNA severely affect its functions, such as replication and transcription, and play a major role in age-related diseases and cancer. A complicated and entangled network of DNA damage response (DDR) mechanisms, including multiple DNA repair pathways, damage tolerance processes, and cell-cycle checkpoints safeguard genomic integrity. Like transcription and replication, DDR is a chromatin-associated process that is generally tightly controlled in time and space. As DNA damag...

  14. DNA-Mediated Electrochemistry

    OpenAIRE

    Gorodetsky, Alon A.; Buzzeo, Marisa C.; Barton, Jacqueline K.

    2008-01-01

    The base pair stack of DNA has been demonstrated as a medium for long-range charge transport chemistry both in solution and at DNA-modified surfaces. This chemistry is exquisitely sensitive to structural perturbations in the base pair stack as occur with lesions, single base mismatches, and protein binding. We have exploited this sensitivity for the development of reliable electrochemical assays based on DNA charge transport at self-assembled DNA monolayers. Here, we discuss the characteristi...

  15. Replicating animal mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Emily A. McKinney

    2013-01-01

    Full Text Available The field of mitochondrial DNA (mtDNA replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading-and lagging-strand synthesis (resembling bacterial genome replication and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS. The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase y, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase. Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.

  16. DNA damage response

    NARCIS (Netherlands)

    G. Giglia-Mari (Giuseppina); A. Zotter (Angelika); W. Vermeulen (Wim)

    2011-01-01

    textabstractStructural changes to DNA severely affect its functions, such as replication and transcription, and play a major role in age-related diseases and cancer. A complicated and entangled network ofDNA damage response (DDR) mechanisms, including multiple DNA repair pathways, damage tolerance p

  17. DNA: Structure and function

    DEFF Research Database (Denmark)

    Sinden, Richard R.; E. Pearson, Christopher; N. Potaman, Vladimir;

    1998-01-01

    This chapter discusses the structure and function of DNA. DNA occupies a critical role in cells, because it is the source of all intrinsic genetic information. Chemically, DNA is a very stable molecule, a characteristic important for a macromolecule that may have to persist in an intact form...

  18. 联合检测AnuA、ANA、抗ds-DNA、ENA在SLE患者中的临床应用%Clinical Application of Combined Detection of AnuA,ANA,anti ds-DNA and anti-ENA in SLE Patients

    Institute of Scientific and Technical Information of China (English)

    罗媛

    2009-01-01

    目的 联合检测SLE患者的多种自身抗体,为临床诊疗提供依据.方法 免疫印迹法检测120例SLE患者AnuA、抗Sm、抗SSA、抗SSB、抗Rib,间接免疫荧光法(IIF)检测抗ds-DNA和ANA,60例健康体检者做为对照组.结果 SLE患者ANA、抗ds-DNA、AunA、抗Sm、抗SSA、抗SSB和抗Rib阳性率分别为69.2%、70.8%、74.2%、35.0%、42.5%、11.7%和26.7%.活动期ANA、抗ds-DNA和AunA阳性率分别为90.7%、98.1%和83.3%,非活动期分别为51.5%、48.5%和66.7%,三项指标差异均有统计学意义(P<0.01、P<0.01和P<0.05). 60例对照组各项检测结果均为阴性.结论 联合检测SLE多种自身抗体在提高SLE的诊断分型及疗效评价等方面具有重要意义.%Objective To combine detection of SLE patients with a variety of autoantibodies, provide a basis for clinical diagnose and treat-ment. Methods AnuA,anti-Sm,anti-SSA,anti-SSB and anti-Rib were determined by western blotting,and anti-ds-DNA and ANA were determined by indirect immunofluorescence(IIF) in 120 cases of SLE patients and 60 controls. Results The positive rates of ANA,anfi ds-DNA, AunA, anti-Sm, anti-SSA, anti-SSB and anti-Rib was 69.2%, 70.8%, 74.2%, 35%, 42.5%, 11.7% and 26.7% in SLE patients. The positive rates of ANA,anti ds-DNA and AunA was 90.7%,98.1% and 83.3% in patients with active stage of SLE. The positive rates of ANA, anti ds-DNA and AunA was 51.5%,48.5% and 66.7% in patients with inactive stage of SLE. Three indicators had statistically signitieance in active stage and inactive stage(P<0.01,P< 0.01 and P< 0.05). All of the test results of 60 controls were negative. Conclusion Combined detection of several autoantibody was important to diagnosis and curative evaluation in SLE.

  19. Detection of rpoB mutations in rifampin-resistant Mycobacterium tuberculosis by use of rolling circle amplification combined with the DNA chip technology%RCA技术联合DNA芯片检测结核分枝杆菌rpoB基因突变

    Institute of Scientific and Technical Information of China (English)

    张江峰; 张亚丽; 曾照芳

    2013-01-01

    目的:应用滚环扩增(rolling circle amplification,RCA)技术以DNA芯片为载体建立一种对结核分枝杆菌耐药基因单碱基突变的快速检测方法.方法:根据结核分枝杆菌利福平耐药rpoB基因序列,设计针对该基因常见突变位点的锁式探针,及固定于基因芯片上的捕获探针.针对临床结核分枝杆菌样本的基因组DNA,PCR扩增含有rpoB基因常见突变位点的特异性DNA片段.将与突变型特异性互补结合并环化的锁式探针与芯片上固定的捕获探针进行杂交,并运用滚环扩增技术,将含有生物素标记的dUTP掺入扩增产物,最后通过与亲和素标记的纳米金反应,并银染增强显色.同时与临床样本的测序结果比较.结果:通过优化反应条件,能特异性的检测出结核分枝杆菌耐利福平rpoB基因的单碱基突变,通过对临床样本的检测,结果与测序结果一致.结论:该方法结合了DNA芯片和滚环扩增技术,能够快速有效的检测出耐药结核的单碱基突变,具有高特异性、高灵敏度.%Objective:To establish a method for rapid detection of single base mutations in resistant Myeobacterium tuberculosis by use of rolling circle amplification combined with the DNA chip technology. Methods According to rpoB gene sequence of Myeobacterium tuberculosis rifampicin resistant, padlock probes of the gene mutation types and the capture probe fixed on the gene chip were designed. Amplification of PCR containing rpoB gene mutation types of specific DNA fragments. Padlock probes via connection and cycclization, hybridization with the capture probe fixed on the gene chip. Using rolling circle amplification doped biotin labeled dUTP into the amplification product, finally through with streptavidin labeled gold nanoparticles reaction, and silver enhancement staining, compared with sequencing. Reults By optimizing the reaction conditions, specific detection of Myeobacterium tuberculosis is rifampicin resistant rpo

  20. Strategies and hurdles using DNA vaccines to fish.

    Science.gov (United States)

    Hølvold, Linn B; Myhr, Anne I; Dalmo, Roy A

    2014-01-01

    DNA vaccinations against fish viral diseases as IHNV at commercial level in Canada against VHSV at experimental level are both success stories. DNA vaccination strategies against many other viral diseases have, however, not yet yielded sufficient results in terms of protection. There is an obvious need to combat many other viral diseases within aquaculture where inactivated vaccines fail. There are many explanations to why DNA vaccine strategies against other viral diseases fail to induce protective immune responses in fish. These obstacles include: 1) too low immunogenicity of the transgene, 2) too low expression of the transgene that is supposed to induce protection, 3) suboptimal immune responses, and 4) too high degradation rate of the delivered plasmid DNA. There are also uncertainties with regard distribution and degradation of DNA vaccines that may have implications for safety and regulatory requirements that need to be clarified. By combining plasmid DNA with different kind of adjuvants one can increase the immunogenicity of the transgene antigen - and perhaps increase the vaccine efficacy. By using molecular adjuvants with or without in combination with targeting assemblies one may expect different responses compared with naked DNA. This includes targeting of DNA vaccines to antigen presenting cells as a central factor in improving their potencies and efficacies by means of encapsulating the DNA vaccine in certain carriers systems that may increase transgene and MHC expression. This review will focus on DNA vaccine delivery, by the use of biodegradable PLGA particles as vehicles for plasmid DNA mainly in fish.

  1. Comparing Charge Transport in Oligonucleotides: RNA:DNA Hybrids and DNA Duplexes.

    Science.gov (United States)

    Li, Yuanhui; Artés, Juan M; Qi, Jianqing; Morelan, Ian A; Feldstein, Paul; Anantram, M P; Hihath, Joshua

    2016-05-19

    Understanding the electronic properties of oligonucleotide systems is important for applications in nanotechnology, biology, and sensing systems. Here the charge-transport properties of guanine-rich RNA:DNA hybrids are compared to double-stranded DNA (dsDNA) duplexes with identical sequences. The conductance of the RNA:DNA hybrids is ∼10 times higher than the equivalent dsDNA, and conformational differences are determined to be the primary reason for this difference. The conductance of the RNA:DNA hybrids is also found to decrease more rapidly than dsDNA when the length is increased. Ab initio electronic structure and Green's function-based density of states calculations demonstrate that these differences arise because the energy levels are more spatially distributed in the RNA:DNA hybrid but that the number of accessible hopping sites is smaller. These combination results indicate that a simple hopping model that treats each individual guanine as a hopping site is insufficient to explain both a higher conductance and β value for RNA:DNA hybrids, and larger delocalization lengths must be considered. PMID:27145167

  2. Counterintuitive DNA Sequence Dependence in Supercoiling-Induced DNA Melting

    NARCIS (Netherlands)

    Vlijm, R.; Torre, J.; Dekker, C.

    2015-01-01

    The metabolism of DNA in cells relies on the balance between hybridized double-stranded DNA (dsDNA) and local de-hybridized regions of ssDNA that provide access to binding proteins. Traditional melting experiments, in which short pieces of dsDNA are heated up until the point of melting into ssDNA, h

  3. Racemic DNA crystallography.

    Science.gov (United States)

    Mandal, Pradeep K; Collie, Gavin W; Kauffmann, Brice; Huc, Ivan

    2014-12-22

    Racemates increase the chances of crystallization by allowing molecular contacts to be formed in a greater number of ways. With the advent of protein synthesis, the production of protein racemates and racemic-protein crystallography are now possible. Curiously, racemic DNA crystallography had not been investigated despite the commercial availability of L- and D-deoxyribo-oligonucleotides. Here, we report a study into racemic DNA crystallography showing the strong propensity of racemic DNA mixtures to form racemic crystals. We describe racemic crystal structures of various DNA sequences and folded conformations, including duplexes, quadruplexes, and a four-way junction, showing that the advantages of racemic crystallography should extend to DNA.

  4. Electronic Transport in DNA

    OpenAIRE

    Klotsa, Daphne; Römer, Rudolf A.; Turner, Matthew S.

    2005-01-01

    We study the electronic properties of DNA by way of a tight-binding model applied to four particular DNA sequences. The charge transfer properties are presented in terms of localisation lengths, crudely speaking the length over which electrons travel. Various types of disorder, including random potentials, are employed to account for different real environments. We have performed calculations on poly(dG)-poly(dC), telomeric-DNA, random-ATGC DNA and lambda-DNA. We find that random and lambda-D...

  5. Fast phylogenetic DNA barcoding

    DEFF Research Database (Denmark)

    Terkelsen, Kasper Munch; Boomsma, Wouter Krogh; Willerslev, Eske;

    2008-01-01

    We present a heuristic approach to the DNA assignment problem based on phylogenetic inferences using constrained neighbour joining and non-parametric bootstrapping. We show that this method performs as well as the more computationally intensive full Bayesian approach in an analysis of 500 insect...... DNA sequences obtained from GenBank. We also analyse a previously published dataset of environmental DNA sequences from soil from New Zealand and Siberia, and use these data to illustrate the fact that statistical approaches to the DNA assignment problem allow for more appropriate criteria...... for determining the taxonomic level at which a particular DNA sequence can be assigned....

  6. Radiation damage to DNA constituents

    International Nuclear Information System (INIS)

    The molecular changes of the DNA molecule, in various systems exposed to inoizing radiation, have been the subject of a great number of studies. In the present work electron spin resonance spectroscopy (ESR) has been applied to irradiated crystalline systems, in particular single crystals of DNA subunits and their derivatives. The main conclusions about the molecular damage are based on this technique in combination with molecular orbital calculations. It should be emphasized that the ESR technique is restricted to damage containing unpaired electrons. These unstable intermediates called free radicals seem, however, to be involved in all molecular models describing the action of radiation on DNA. One of the premises for a detailed theory of the radiation induced reactions at the physico-chemical level seems to involve exact knowledge of the induced free radicals as well as the modes of their formation and fate. For DNA, as such, it is hardly possible to arrive at such a level of knowledge since the molecular complexity prevents selective studies of the many different radiation induced products. One possible approach is to study the free radicals formed in the constituents of DNA. In the present work three lines of approach should be mentioned. The first is based on the observation that radical formation in general causes only minor structural alterations to the molecule in question. The use of isotopes with different spin and magnetic moment (in particular deuterium) may also serve a source of information. Deuteration leads to a number of protons, mainly NH - and OH, becoming substituted, and if any of these are involved in interactions with unpaired protons the resonance pattern is influeneed. The third source of information is molecular orbital calculation. The electron spin density distribution is a function in the three dimensional space based on the system's electronic wave functions. This constitutes the basis for the idea that ESR data can be correlated with

  7. Diagnostic Value Analysis about Anti-dsDNA Antibody, Anti-Smith Antibody and Anti-Rib-P Antibody of Systemic Lupus Erythematosus by Protein Chip Technology Combined Detection%蛋白芯片技术联合检测抗dsDNA抗体、抗Smith抗体和抗Rib-P抗体对SLE诊断价值分析

    Institute of Scientific and Technical Information of China (English)

    杨艳丽; 任健康; 李亚娥; 胡淑玲

    2012-01-01

    Objective To evaluate the combined detection of the anti-dsDNA antibody .anti-Smith antibody and anti-Rib-P antibody in protein chip technique for the diagnosis of systemic lupus erythematosus (SLE). Methods Detected the anti-ds DNA antibody.anti-Smith antibody and anti-Rib-P antibody in the serum of 85 SLE patients,129 patients with other autoimmune diseases and 43 healthy subjects by applying protein-chip technology. Results The positive rate(50. 59% ,32. 94% and 37. 65%) of anti-dsDNA antibody,anti-Smith antibody and anti-Rib-P antibody in SLE patients' serum and the positive rate (78. 82%) of their combined detection was significantly higher than that of the disease control group. There was difference (χ2:100. 269,52. 36,62.481 and 155. 51, P<0. 01). The normal control group was all negative. Among SLE patients,for the anti-dsDNA antibody,the sensitivity was 50. 59% and specificity was 99. 22%,for the anti-Smith antibody, the sensitivity was 32. 94% and specificity was 97. 67%;for the anti-Rib-P antibody, the sensitivity was 37.65% and specificity was 97. 67% ;for the three autoantibodies' combined detection, the sensitivity was 78. 82% and specificity was 94. 57%. The sensitivity of combined detection was significantly higher than that of every single detection. Conclusion As the important SLE antibodies,anti-dsDNA antibody, anti-Smith antibody and anti-Rib-P antibody with high specificity can complement with each other in the simultaneous detection on a protein chip. It improve the sensitivity and efficiency of SLE diagnosis, reduce the missed diagnosis and misdiagnosis of SLE diagnosis, so it is important for SLE diagnosis,treatment and prognosis.%目的 评价蛋白芯片技术联合检测抗dsDNA抗体、抗Smith抗体和抗Rib-P抗体对于系统性红斑狼疮(SLE)诊断的价值.方法 采用蛋白芯片技术对85例SLE患者、129例其他自身免疫性疾病患者和43例健康体检者血清中的抗dsDNA抗体、抗Smith抗体和

  8. DNA in Nanoscale Electronics

    Science.gov (United States)

    Slinker, Jason

    2012-10-01

    DNA, the quintessential molecule of life, possesses a number of attractive properties for use in nanoscale circuits. Charge transport (CT) through DNA itself is of both fundamental and practical interest. Fundamentally, DNA has a unique configuration of π-stacked bases in a well ordered, double helical structure. Given its unparalleled importance to life processes and its arrangement of conjugated subunits, DNA has been a compelling target of conductivity studies. In addition, further understanding of DNA CT will elucidate the biological implications of this process and advance its use in sensing technologies. We have investigated the fundamentals of DNA CT by measuring the electrochemistry of DNA monolayers under biologically-relevant conditions. We have uncovered both fundamental kinetic parameters to distinguish between competing models of operation as well as the practical implications of DNA CT for sensing. Furthermore, we are leveraging our studies of DNA conductivity for the manufacture of nanoscale circuits. We are investigating the electrical properties and self-assembly of DNA nanowires containing artificial base pair surrogates, which can be prepared through low cost and high throughput automated DNA synthesis. This unique and economically viable approach will establish a new paradigm for the scalable manufacture of nanoscale semiconductor devices.

  9. Biophysics of DNA

    CERN Document Server

    Vologodskii, Alexander

    2015-01-01

    Surveying the last sixty years of research, this book describes the physical properties of DNA in the context of its biological functioning. It is designed to enable both students and researchers of molecular biology, biochemistry and physics to better understand the biophysics of DNA, addressing key questions and facilitating further research. The chapters integrate theoretical and experimental approaches, emphasising throughout the importance of a quantitative knowledge of physical properties in building and analysing models of DNA functioning. For example, the book shows how the relationship between DNA mechanical properties and the sequence specificity of DNA-protein binding can be analyzed quantitatively by using our current knowledge of the physical and structural properties of DNA. Theoretical models and experimental methods in the field are critically considered to enable the reader to engage effectively with the current scientific literature on the physical properties of DNA.

  10. The application of HPV-DNA detection combined with VIA test in screening of precancerous lesion of uterine cervix%高危型HPV检测联合肉眼醋酸试验在宫颈癌前病变筛查中的应用

    Institute of Scientific and Technical Information of China (English)

    赵金娟

    2012-01-01

    目的:研究在宫颈癌前病变筛查中采用高危型HPV检测联合肉眼醋酸试验的应用效果.方法:选取2007年8月~2010年2月于我院治疗的可疑宫颈癌前病变患者532例,应用5%醋酸和Lugol's 液涂抹于宫颈表面,筛选出有异常着色的患者320例,再采用实时荧光定量PCR技术检测高危型HPV病毒感染情况.结果:320例肉眼醋酸试验异常着色患者进行高危型HPV病毒感染检测发现,HPV阳性感染190例,阳性率为59.38%;212例肉眼醋酸试验正常着色患者进行高危型HPV病毒感染检测发现,HPV阳性感染18例,阳性率为8.49%.结论:肉眼醋酸试验联合高危型HPV检测可及时、有效地发现宫颈癌前病变,可作为宫颈癌前病变筛查方案应用于临床.%Objective: To study the efficiency of HPV-DNA detection combined with visual inspection with acetic acid (VIA) test in screening of precancerous lesions of uterine cervix. Methods: 532 suspected precancerous lesion of uterine cervix patients in our hospital from August 2007 to February 2010 were selected. 5% acetic acid and Lugol's liquid were painted on the surface of cervix of all patients, and 320 cases of patients were screened by the abnormal color. Then the re -al-time fluorescence quantitative polymerase chain reaction technology was used to test high risk HPV infection in all pa -tients. Results: High risk HPV infection test of 320 patients who were showed abnormal color in VIA test was showed that 190 cases got HPV positive infection, the positive rate was 59.38%. High risk HPV infection test of 212 patients who were showed normal color in VIA test was showed that 18 cases got HPV positive infection, the positive rate was 8.49%. Conclusion: VIA test combined with HPV-DNA checking can timely and effectively discover precancerous lesion, which can be used in clinic as precancerous lesion screening plan.

  11. ELISA-like Analysis of Cisplatinated DNA Using Magnetic Separation

    OpenAIRE

    Kristyna Smerkova; Marcela Vlcnovska; Simona Dostalova; Vedran Milosavljevic; Pavel Kopel; Tomas Vaculovic; Sona Krizkova; Marketa Vaculovicova; Vojtech Adam; Rene Kizek

    2015-01-01

    Cisplatin belongs to the most widely used cytostatic drugs. The determination of the presence of the DNA-cisplatin adducts may not only signal the guanine-rich regions but also monitor the interaction reaction between DNA and the drug in terms of speed of interaction. In this work, the combined advantages of magnetic particles-based isolation/purification with fluorescent properties of quantum dots (QDs) and antibodies targeted on specific recognition of DNA-cisplatin adducts are demonstra...

  12. The short-term therapeutic effects of DNA immunoadsorbent combined with medication on patients with ;severe systemic lupus erythematosus%DNA免疫吸附联合药物治疗重度系统性红斑狼疮的近期疗效观察

    Institute of Scientific and Technical Information of China (English)

    吴潮清; 黄玲; 唐业莹; 熊礼佳; 贺红光; 刘芸芳; 李瑾瑜; 王浩宇; 唐盛; 叶琨; 彭小梅

    2014-01-01

    Objective To evaluate the short-term therapeutic effects of DNA immunoadsorbent (IA) combined with glucocorticoid and immune depressant on patients with severe systemic lupus erythematosus(SLE). Methods 32 patients with severe SLE were selected to undergo DNA IA treatment combined with glucocorticoid plus cyclophosphamide therapy, and each patient received IA therapy 3 times, once 2.5 hours, with an interval of 24-48 hours to take another two times of IA. The changes in SLE disease activity index(SLEDAI)score, health status evaluation indexes〔 physiologic functional( PF) and emotional health( MH) scores〕,renal function indexes〔 blood urea nitrogen(BUN)and serum creatinine(SCr)〕 were observed; and anti-double stranded DNA antibody( ds-DNA), immunoglobulin (IgA, IgG, IgM), complements(C3 and C4)and high-sensitivity C-reactive protein (hs-CRP) were examined before and after IA treatment for 2 weeks. Results Two weeks after the combination therapy, the SLEDAI score, BUN, SCr, dsDNA, IgA, IgG, IgM, hs-CRP were significantly lower than those before treatment 〔SLEDAI score : 14.38±3.85 vs. 15.69±1.40, BUN (mmol/L): 11.22±4.78 vs. 16.31±7.90, SCr (μmol/L): 127.02±38.17 vs. 167.25±45.63, dsDNA( U/L): 1.36±0.12 vs. 1.43±0.18, IgA( g/L): 2.41±0.73 vs. 2.59±0.86, IgG( g/L): 16.82±4.83 vs. 21.01±4.84, IgM( g/L): 1.64±0.45 vs. 1.75±0.58, hs-CRP( mg/L): 14.41±2.20 vs. 14.94±2.60, P<0.05 or P<0.01〕; PF score, MH score, complement C3 were increased〔 PF score : 71.19±17.53 vs. 56.66±22.41, MH score : 74.01±15.72 vs. 61.50±17.98, C3( g/L): 0.56±0.09 vs. 0.52±0.10, all P<0.05〕; clinical symptoms were improved significantly, and no significant adverse reactions were found. Conclusion IA combined with medical treatment has shown that it has significant therapeutic effect for treatment of patients with severe SLE, and it may decrease the levels of dsDNA, IgA, IgG, IgM,hs-CRP, and increase the level of complement C3.%目的:观察DNA免疫吸附

  13. Improving DNA vaccine performance through vector design.

    Science.gov (United States)

    Williams, James A

    2014-01-01

    DNA vaccines are a rapidly deployed next generation vaccination platform for treatment of human and animal disease. DNA delivery devices, such as electroporation and needle free jet injectors, are used to increase gene transfer. This results in higher antigen expression which correlates with improved humoral and cellular immunity in humans and animals. This review highlights recent vector and transgene design innovations that improve DNA vaccine performance. These new vectors improve antigen expression, increase plasmid manufacturing yield and quality in bioreactors, and eliminate antibiotic selection and other potential safety issues. A flowchart for designing synthetic antigen transgenes, combining antigen targeting, codon-optimization and bioinformatics, is presented. Application of improved vectors, of antibiotic free plasmid production, and cost effective manufacturing technologies will be critical to ensure safety, efficacy, and economically viable manufacturing of DNA vaccines currently under development for infectious disease, cancer, autoimmunity, immunotolerance and allergy indications.

  14. A DNA barcode for land plants.

    Science.gov (United States)

    2009-08-01

    DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF-atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK-psbI spacer, and trnH-psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants. PMID:19666622

  15. A DNA barcode for land plants

    Science.gov (United States)

    Hollingsworth, Peter M.; Forrest, Laura L.; Spouge, John L.; Hajibabaei, Mehrdad; Ratnasingham, Sujeevan; van der Bank, Michelle; Chase, Mark W.; Cowan, Robyn S.; Erickson, David L.; Fazekas, Aron J.; Graham, Sean W.; James, Karen E.; Kim, Ki-Joong; Kress, W. John; Schneider, Harald; van AlphenStahl, Jonathan; Barrett, Spencer C.H.; van den Berg, Cassio; Bogarin, Diego; Burgess, Kevin S.; Cameron, Kenneth M.; Carine, Mark; Chacón, Juliana; Clark, Alexandra; Clarkson, James J.; Conrad, Ferozah; Devey, Dion S.; Ford, Caroline S.; Hedderson, Terry A.J.; Hollingsworth, Michelle L.; Husband, Brian C.; Kelly, Laura J.; Kesanakurti, Prasad R.; Kim, Jung Sung; Kim, Young-Dong; Lahaye, Renaud; Lee, Hae-Lim; Long, David G.; Madriñán, Santiago; Maurin, Olivier; Meusnier, Isabelle; Newmaster, Steven G.; Park, Chong-Wook; Percy, Diana M.; Petersen, Gitte; Richardson, James E.; Salazar, Gerardo A.; Savolainen, Vincent; Seberg, Ole; Wilkinson, Michael J.; Yi, Dong-Keun; Little, Damon P.

    2009-01-01

    DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants. PMID:19666622

  16. Multiplexed Sequence Encoding: A Framework for DNA Communication.

    Science.gov (United States)

    Zakeri, Bijan; Carr, Peter A; Lu, Timothy K

    2016-01-01

    Synthetic DNA has great propensity for efficiently and stably storing non-biological information. With DNA writing and reading technologies rapidly advancing, new applications for synthetic DNA are emerging in data storage and communication. Traditionally, DNA communication has focused on the encoding and transfer of complete sets of information. Here, we explore the use of DNA for the communication of short messages that are fragmented across multiple distinct DNA molecules. We identified three pivotal points in a communication-data encoding, data transfer & data extraction-and developed novel tools to enable communication via molecules of DNA. To address data encoding, we designed DNA-based individualized keyboards (iKeys) to convert plaintext into DNA, while reducing the occurrence of DNA homopolymers to improve synthesis and sequencing processes. To address data transfer, we implemented a secret-sharing system-Multiplexed Sequence Encoding (MuSE)-that conceals messages between multiple distinct DNA molecules, requiring a combination key to reveal messages. To address data extraction, we achieved the first instance of chromatogram patterning through multiplexed sequencing, thereby enabling a new method for data extraction. We envision these approaches will enable more widespread communication of information via DNA. PMID:27050646

  17. DNA-nanostructure-assembly by sequential spotting

    Directory of Open Access Journals (Sweden)

    Breitenstein Michael

    2011-11-01

    Full Text Available Abstract Background The ability to create nanostructures with biomolecules is one of the key elements in nanobiotechnology. One of the problems is the expensive and mostly custom made equipment which is needed for their development. We intended to reduce material costs and aimed at miniaturization of the necessary tools that are essential for nanofabrication. Thus we combined the capabilities of molecular ink lithography with DNA-self-assembling capabilities to arrange DNA in an independent array which allows addressing molecules in nanoscale dimensions. Results For the construction of DNA based nanostructures a method is presented that allows an arrangement of DNA strands in such a way that they can form a grid that only depends on the spotted pattern of the anchor molecules. An atomic force microscope (AFM has been used for molecular ink lithography to generate small spots. The sequential spotting process allows the immobilization of several different functional biomolecules with a single AFM-tip. This grid which delivers specific addresses for the prepared DNA-strand serves as a two-dimensional anchor to arrange the sequence according to the pattern. Once the DNA-nanoarray has been formed, it can be functionalized by PNA (peptide nucleic acid to incorporate advanced structures. Conclusions The production of DNA-nanoarrays is a promising task for nanobiotechnology. The described method allows convenient and low cost preparation of nanoarrays. PNA can be used for complex functionalization purposes as well as a structural element.

  18. Targeting DNA Replication Stress for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Jun Zhang

    2016-08-01

    Full Text Available The human cellular genome is under constant stress from extrinsic and intrinsic factors, which can lead to DNA damage and defective replication. In normal cells, DNA damage response (DDR mediated by various checkpoints will either activate the DNA repair system or induce cellular apoptosis/senescence, therefore maintaining overall genomic integrity. Cancer cells, however, due to constitutive growth signaling and defective DDR, may exhibit “replication stress” —a phenomenon unique to cancer cells that is described as the perturbation of error-free DNA replication and slow-down of DNA synthesis. Although replication stress has been proven to induce genomic instability and tumorigenesis, recent studies have counterintuitively shown that enhancing replicative stress through further loosening of the remaining checkpoints in cancer cells to induce their catastrophic failure of proliferation may provide an alternative therapeutic approach. In this review, we discuss the rationale to enhance replicative stress in cancer cells, past approaches using traditional radiation and chemotherapy, and emerging approaches targeting the signaling cascades induced by DNA damage. We also summarize current clinical trials exploring these strategies and propose future research directions including the use of combination therapies, and the identification of potential new targets and biomarkers to track and predict treatment responses to targeting DNA replication stress.

  19. DNA hydration studied by neutron fiber diffraction

    Energy Technology Data Exchange (ETDEWEB)

    Fuller, W.; Forsyth, V.T.; Mahendrasingam, A.; Langan, P.; Pigram, W.J. [Keele Univ. (United Kingdom)] [and others

    1994-12-31

    The development of neutron high angle fiber diffraction to investigate the location of water around the deoxyribonucleic acid (DNA) double-helix is described. The power of the technique is illustrated by its application to the D and A conformations of DNA using the single crystal diffractometer, D19, at the Institute Laue-Langevin, Grenoble and the time of flight diffractometer, SXD, at the Rutherford Appleton ISIS Spallation Neutron Source. These studies show the existence of bound water closely associated with the DNA. The patterns of hydration in these two DNA conformations are quite distinct and are compared to those observed in X-ray single crystal studies of two-stranded oligodeoxynucleotides. Information on the location of water around the DNA double-helix from the neutron fiber diffraction studies is combined with that on the location of alkali metal cations from complementary X-ray high angle fiber diffraction studies at the Daresbury Laboratory SRS using synchrotron radiation. These analyses emphasize the importance of viewing DNA, water and ions as a single system with specific interactions between the three components and provide a basis for understanding the effect of changes in the concentration of water and ions in inducing conformations] transitions in the DNA double-helix.

  20. Targeting DNA Replication Stress for Cancer Therapy

    Science.gov (United States)

    Zhang, Jun; Dai, Qun; Park, Dongkyoo; Deng, Xingming

    2016-01-01

    The human cellular genome is under constant stress from extrinsic and intrinsic factors, which can lead to DNA damage and defective replication. In normal cells, DNA damage response (DDR) mediated by various checkpoints will either activate the DNA repair system or induce cellular apoptosis/senescence, therefore maintaining overall genomic integrity. Cancer cells, however, due to constitutive growth signaling and defective DDR, may exhibit “replication stress” —a phenomenon unique to cancer cells that is described as the perturbation of error-free DNA replication and slow-down of DNA synthesis. Although replication stress has been proven to induce genomic instability and tumorigenesis, recent studies have counterintuitively shown that enhancing replicative stress through further loosening of the remaining checkpoints in cancer cells to induce their catastrophic failure of proliferation may provide an alternative therapeutic approach. In this review, we discuss the rationale to enhance replicative stress in cancer cells, past approaches using traditional radiation and chemotherapy, and emerging approaches targeting the signaling cascades induced by DNA damage. We also summarize current clinical trials exploring these strategies and propose future research directions including the use of combination therapies, and the identification of potential new targets and biomarkers to track and predict treatment responses to targeting DNA replication stress. PMID:27548226

  1. Charting the Structure and Energetics of Packaged DNA in Bacteriophages

    Science.gov (United States)

    Qiu, Xiangyun; Rau, Donald C.; Parsegian, V. Adrian; Fang, Li Tai; Knobler, Charles M.; Gelbart, William M.

    2009-03-01

    Many bacterial viruses resort to pressure in order to infect bacteria, e.g., lambda phage stores its dsDNA genome at surprisingly high pressure and then uses this pressure to drive delivery of the genome. We report on a biophysical interrogation of the DNA configuration and pressure in lambda phage by combining structural and thermodynamic measurements with theoretical modeling. Changes in DNA organization in the capsid are monitored using solution small angle x-ray scattering (SAXS). We vary the DNA-DNA repulsion and DNA bending contributions to the capsid pressure by changing salt concentrations and packaged length, and augment SAXS data with osmotic stress measurements to elicit the evolving structure and energetics of the packaged DNA.

  2. DNA nanotechnology from the test tube to the cell

    Science.gov (United States)

    Chen, Yuan-Jyue; Groves, Benjamin; Muscat, Richard A.; Seelig, Georg

    2015-09-01

    The programmability of Watson-Crick base pairing, combined with a decrease in the cost of synthesis, has made DNA a widely used material for the assembly of molecular structures and dynamic molecular devices. Working in cell-free settings, researchers in DNA nanotechnology have been able to scale up system complexity and quantitatively characterize reaction mechanisms to an extent that is infeasible for engineered gene circuits or other cell-based technologies. However, the most intriguing applications of DNA nanotechnology -- applications that best take advantage of the small size, biocompatibility and programmability of DNA-based systems -- lie at the interface with biology. Here, we review recent progress in the transition of DNA nanotechnology from the test tube to the cell. We highlight key successes in the development of DNA-based imaging probes, prototypes of smart therapeutics and drug delivery systems, and explore the future challenges and opportunities for cellular DNA nanotechnology.

  3. Geant4-DNA: overview and recent developments

    Science.gov (United States)

    Štěpán, Václav

    Space travel and high altitude flights are inherently associated with prolonged exposure to cosmic and solar radiation. Understanding and simulation of radiation action on cellular and subcellular level contributes to precise assessment of the associated health risks and remains a challenge of today’s radiobiology research. The Geant4-DNA project (http://geant4-dna.org) aims at developing an experimentally validated simulation platform for modelling of the damage induced by ionizing radiation at DNA level. The platform is based on the Geant4 Monte Carlo simulation toolkit. This project extends specific functionalities of Geant4 in following areas: The step-by-step single scattering modelling of elementary physical interactions of electrons, protons, alpha particles and light ions with liquid water and DNA bases, for the so-called “physical” stage. The modelling of the “physico-chemical and chemical” stages corresponding to the production, the diffusion, the chemical reactions occurring between chemical species produced by water radiolysis, and to the radical attack on the biological targets. Physical and chemical stage simulations are combined with biological target models on several scales, from DNA double helix, through nucleosome, to chromatin segments and cell geometries. In addition, data mining clustering algorithms have been developed and optimised for the purpose of DNA damage scoring in simulated tracks. Experimental measurements on pBR322 plasmid DNA are being carried out in order to validate the Geant4-DNA models. The plasmid DNA has been irradiated in dry conditions by protons with energies from 100 keV to 30 MeV and in aqueous conditions, with and without scavengers, by 30 MeV protons, 290 MeV/u carbon and 500 MeV/u iron ions. Agarose gel electrophoresis combined with enzymatic treatment has been used to measure the resulting DNA damage. An overview of the developments undertaken by the Geant4-DNA collaboration including a description of

  4. Forensic DNA analysis.

    Science.gov (United States)

    McDonald, Jessica; Lehman, Donald C

    2012-01-01

    Before the routine use of DNA profiling, blood typing was an important forensic tool. However, blood typing was not very discriminating. For example, roughly 30% of the United States population has type A-positive blood. Therefore, if A-positive blood were found at a crime scene, it could have come from 30% of the population. DNA profiling has a much better ability for discrimination. Forensic laboratories no longer routinely determine blood type. If blood is found at a crime scene, DNA profiling is performed. From Jeffrey's discovery of DNA fingerprinting to the development of PCR of STRs to the formation of DNA databases, our knowledge of DNA and DNA profiling have expanded greatly. Also, the applications for which we use DNA profiling have increased. DNA profiling is not just used for criminal case work, but it has expanded to encompass paternity testing, disaster victim identification, monitoring bone marrow transplants, detecting fetal cells in a mother's blood, tracing human history, and a multitude of other areas. The future of DNA profiling looks expansive with the development of newer instrumentation and techniques. PMID:22693781

  5. The binding of in vitro synthesized adenovirus DNA binding protein to single-stranded DNA is stimulated by zinc ions

    NARCIS (Netherlands)

    Vos, H.L.; Lee, F.M. van der; Sussenbach, J.S.

    1988-01-01

    We have synthesized wild type DNA binding protein (DBP) of adenovirus type 5 (Ad5) and several truncated forms of this protein by a combination of in vitro transcription and translation. The proteins obtained were tested for binding to a single-stranded DNA-cellulose column. It could be shown that f

  6. DNA profiles from fingermarks.

    Science.gov (United States)

    Templeton, Jennifer E L; Linacre, Adrian

    2014-11-01

    Criminal investigations would be considerably improved if DNA profiles could be routinely generated from single fingermarks. Here we report a direct DNA profiling method that was able to generate interpretable profiles from 71% of 170 fingermarks. The data are based on fingermarks from all 5 digits of 34 individuals. DNA was obtained from the fingermarks using a swab moistened with Triton-X, and the fibers were added directly to one of two commercial DNA profiling kits. All profiles were obtained without increasing the number of amplification cycles; therefore, our method is ideally suited for adoption by the forensic science community. We indicate the use of the technique in a criminal case in which a DNA profile was generated from a fingermark on tape that was wrapped around a drug seizure. Our direct DNA profiling approach is rapid and able to generate profiles from touched items when current forensic practices have little chance of success. PMID:25391915

  7. Electrocatalysis in DNA Sensors.

    Science.gov (United States)

    Furst, Ariel; Hill, Michael G; Barton, Jacqueline K

    2014-12-14

    Electrocatalysis is often thought of solely in the inorganic realm, most often applied to energy conversion in fuel cells. However, the ever-growing field of bioelectrocatalysis has made great strides in advancing technology for both biofuel cells as well as biological detection platforms. Within the context of bioelectrocatalytic detection systems, DNA-based platforms are especially prevalent. One subset of these platforms, the one we have developed, takes advantage of the inherent charge transport properties of DNA. Electrocatalysis coupled with DNA-mediated charge transport has enabled specific and sensitive detection of lesions, mismatches and DNA-binding proteins. Even greater signal amplification from these platforms is now being achieved through the incorporation of a secondary electrode to the platform both for patterning DNA arrays and for detection. Here, we describe the evolution of this new DNA sensor technology. PMID:25435647

  8. DNA replication and cancer

    DEFF Research Database (Denmark)

    Boyer, Anne-Sophie; Walter, David; Sørensen, Claus Storgaard

    2016-01-01

    A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways...... to promote genome integrity during DNA replication. This includes suppressing new replication origin firing, stabilization of replicating forks, and the safe restart of forks to prevent any loss of genetic information. Here, we describe mechanisms by which oncogenes can interfere with DNA replication thereby...... causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy....

  9. Tracking earthworm communities from soil DNA.

    Science.gov (United States)

    Bienert, Friederike; De Danieli, Sébastien; Miquel, Christian; Coissac, Eric; Poillot, Carole; Brun, Jean-Jacques; Taberlet, Pierre

    2012-04-01

    Earthworms are known for their important role within the functioning of an ecosystem, and their diversity can be used as an indicator of ecosystem health. To date, earthworm diversity has been investigated through conventional extraction methods such as handsorting, soil washing or the application of a mustard solution. Such techniques are time consuming and often difficult to apply. We showed that combining DNA metabarcoding and next-generation sequencing facilitates the identification of earthworm species from soil samples. The first step of our experiments was to create a reference database of mitochondrial DNA (mtDNA) 16S gene for 14 earthworm species found in the French Alps. Using this database, we designed two new primer pairs targeting very short and informative DNA sequences (about 30 and 70 bp) that allow unambiguous species identification. Finally, we analysed extracellular DNA taken from soil samples in two localities (two plots per locality and eight samples per plot). The two short metabarcode regions led to the identification of a total of eight earthworm species. The earthworm communities identified by the DNA-based approach appeared to be well differentiated between the two localities and are consistent with results derived from inventories collected using the handsorting method. The possibility of assessing earthworm communities from hundreds or even thousands of localities through the use of extracellular soil DNA will undoubtedly stimulate further ecological research on these organisms. Using the same DNA extracts, our study also illustrates the potential of environmental DNA as a tool to assess the diversity of other soil-dwelling animal taxa.

  10. Hysteresis in pressure-driven DNA denaturation.

    Directory of Open Access Journals (Sweden)

    Enrique Hernández-Lemus

    Full Text Available In the past, a great deal of attention has been drawn to thermal driven denaturation processes. In recent years, however, the discovery of stress-induced denaturation, observed at the one-molecule level, has revealed new insights into the complex phenomena involved in the thermo-mechanics of DNA function. Understanding the effect of local pressure variations in DNA stability is thus an appealing topic. Such processes as cellular stress, dehydration, and changes in the ionic strength of the medium could explain local pressure changes that will affect the molecular mechanics of DNA and hence its stability. In this work, a theory that accounts for hysteresis in pressure-driven DNA denaturation is proposed. We here combine an irreversible thermodynamic approach with an equation of state based on the Poisson-Boltzmann cell model. The latter one provides a good description of the osmotic pressure over a wide range of DNA concentrations. The resulting theoretical framework predicts, in general, the process of denaturation and, in particular, hysteresis curves for a DNA sequence in terms of system parameters such as salt concentration, density of DNA molecules and temperature in addition to structural and configurational states of DNA. Furthermore, this formalism can be naturally extended to more complex situations, for example, in cases where the host medium is made up of asymmetric salts or in the description of the (helical-like charge distribution along the DNA molecule. Moreover, since this study incorporates the effect of pressure through a thermodynamic analysis, much of what is known from temperature-driven experiments will shed light on the pressure-induced melting issue.

  11. Repair of DNA-containing pyrimidine dimers

    International Nuclear Information System (INIS)

    Ultraviolet light-induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. The most direct biochemical mechanism responding to this kind of genotoxicity involves direct photoreversal by flavin enzymes that specifically monomerize pyrimidine:pyrimidine dimers monophotonically in the presence of visible light. Incision reactions are catalyzed by a combined pyrimidine dimer DNA-glycosylase:apyrimidinic endonuclease found in some highly UV-resistant organisms. At a higher level of complexity, Escherichia coli has a uvr DNA repair system comprising the UvrA, UvrB, and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway, which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding is associated with localized topological unwinding of DNA. This same protein complex can catalyze an ATPase-dependent 5'----3'-directed strand displacement of D-loop DNA or short single strands annealed to a single-stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled excision-resynthesis step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress-induced protease that also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA, it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.21 references

  12. Repair of DNA-containing pyrimidine dimers

    Energy Technology Data Exchange (ETDEWEB)

    Grossman, L.; Caron, P.R.; Mazur, S.J.; Oh, E.Y.

    1988-08-01

    Ultraviolet light-induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. The most direct biochemical mechanism responding to this kind of genotoxicity involves direct photoreversal by flavin enzymes that specifically monomerize pyrimidine:pyrimidine dimers monophotonically in the presence of visible light. Incision reactions are catalyzed by a combined pyrimidine dimer DNA-glycosylase:apyrimidinic endonuclease found in some highly UV-resistant organisms. At a higher level of complexity, Escherichia coli has a uvr DNA repair system comprising the UvrA, UvrB, and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway, which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding is associated with localized topological unwinding of DNA. This same protein complex can catalyze an ATPase-dependent 5'----3'-directed strand displacement of D-loop DNA or short single strands annealed to a single-stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled excision-resynthesis step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress-induced protease that also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA, it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.21 references.

  13. Tracking earthworm communities from soil DNA.

    Science.gov (United States)

    Bienert, Friederike; De Danieli, Sébastien; Miquel, Christian; Coissac, Eric; Poillot, Carole; Brun, Jean-Jacques; Taberlet, Pierre

    2012-04-01

    Earthworms are known for their important role within the functioning of an ecosystem, and their diversity can be used as an indicator of ecosystem health. To date, earthworm diversity has been investigated through conventional extraction methods such as handsorting, soil washing or the application of a mustard solution. Such techniques are time consuming and often difficult to apply. We showed that combining DNA metabarcoding and next-generation sequencing facilitates the identification of earthworm species from soil samples. The first step of our experiments was to create a reference database of mitochondrial DNA (mtDNA) 16S gene for 14 earthworm species found in the French Alps. Using this database, we designed two new primer pairs targeting very short and informative DNA sequences (about 30 and 70 bp) that allow unambiguous species identification. Finally, we analysed extracellular DNA taken from soil samples in two localities (two plots per locality and eight samples per plot). The two short metabarcode regions led to the identification of a total of eight earthworm species. The earthworm communities identified by the DNA-based approach appeared to be well differentiated between the two localities and are consistent with results derived from inventories collected using the handsorting method. The possibility of assessing earthworm communities from hundreds or even thousands of localities through the use of extracellular soil DNA will undoubtedly stimulate further ecological research on these organisms. Using the same DNA extracts, our study also illustrates the potential of environmental DNA as a tool to assess the diversity of other soil-dwelling animal taxa. PMID:22250728

  14. Quantitative radiommunoassay for DNA-binding antibodies

    International Nuclear Information System (INIS)

    A radioimmunoassay (RIA) is described for the measurement of serum immunoglobulins capable of binding to double-standard or single-standard DNA. DNA attached to Sephadex G-50 by ultraviolet radiation was used as a solid- phase immunoabsorbent for DNA-binding proteins from serum. Goat anti-human (GAH) IgG (125I-labeled) were used to detect the human immunoglobulins bound onto the washed DNA-Sephadex. The quantities of immunoglobulins bound were determined by comparison with a standard curve constructed by dilution of a plasma from an systemic lupus erythematosus (SLE) patient containing known amounts of bound, DNA-specific IgM and IgG. Another RIA was employed for measuring levels of IgG and IgM. In combination with measurements of the total serum IgM and IgG, the RIA allowed for the determination of the fraction of the total serum IgM or IgG that was specific for double- or single-standard DNA. For a pool of normal human sera the quantities were as follows: 0.04% of the total IgM and 0.001% of the total IgG bound double-standard DNA; 0.22% of the total IgM and 0.05% of the total IgG bound single-stranded DNA. This capability is important because information regarding the quantitative measurement of antibodies to DNA and their class determination may be of significance in monitoring the status of subjects with SLE

  15. Binary electrokinetic separation of target DNA from background DNA primers.

    Energy Technology Data Exchange (ETDEWEB)

    James, Conrad D.; Derzon, Mark Steven

    2005-10-01

    This report contains the summary of LDRD project 91312, titled ''Binary Electrokinetic Separation of Target DNA from Background DNA Primers''. This work is the first product of a collaboration with Columbia University and the Northeast BioDefense Center of Excellence. In conjunction with Ian Lipkin's lab, we are developing a technique to reduce false positive events, due to the detection of unhybridized reporter molecules, in a sensitive and multiplexed detection scheme for nucleic acids developed by the Lipkin lab. This is the most significant problem in the operation of their capability. As they are developing the tools for rapidly detecting the entire panel of hemorrhagic fevers this technology will immediately serve an important national need. The goal of this work was to attempt to separate nucleic acid from a preprocessed sample. We demonstrated the preconcentration of kilobase-pair length double-stranded DNA targets, and observed little preconcentration of 60 base-pair length single-stranded DNA probes. These objectives were accomplished in microdevice formats that are compatible with larger detection systems for sample pre-processing. Combined with Columbia's expertise, this technology would enable a unique, fast, and potentially compact method for detecting/identifying genetically-modified organisms and multiplexed rapid nucleic acid identification. Another competing approach is the DARPA funded IRIS Pharmaceutical TIGER platform which requires many hours for operation, and an 800k$ piece of equipment that fills a room. The Columbia/SNL system could provide a result in 30 minutes, at the cost of a few thousand dollars for the platform, and would be the size of a shoebox or smaller.

  16. DNA Microarray Technique

    Directory of Open Access Journals (Sweden)

    Thakare SP

    2012-11-01

    Full Text Available DNA Microarray is the emerging technique in Biotechnology. The many varieties of DNA microarray or DNA chip devices and systems are described along with their methods for fabrication and their use. It also includes screening and diagnostic applications. The DNA microarray hybridization applications include the important areas of gene expression analysis and genotyping for point mutations, single nucleotide polymorphisms (SNPs, and short tandem repeats (STRs. In addition to the many molecular biological and genomic research uses, this review covers applications of microarray devices and systems for pharmacogenomic research and drug discovery, infectious and genetic disease and cancer diagnostics, and forensic and genetic identification purposes.

  17. The Bacillus subtilis DnaD and DnaB Proteins Exhibit Different DNA Remodelling Activities

    OpenAIRE

    Zhang, Wenke; Carneiro, Maria J. V. M.; Turner, Ian J.; ALLEN, Stephanie; Roberts, Clive J.; Soultanas, Panos

    2005-01-01

    Primosomal protein cascades load the replicative helicase onto DNA. In Bacillus subtilis a putative primosomal cascade involving the DnaD-DnaB-DnaI proteins has been suggested to participate in both the DnaA and PriA-dependent loading of the replicative helicase DnaC onto the DNA. Recently we discovered that DnaD has a global remodelling DNA activity suggesting a more widespread role in bacterial nucleoid architecture. Here, we show that DnaB forms a “square-like” tetramer with a hole in the ...

  18. Structural stability versus conformational sampling in biomolecular systems: Why is the charge transfer efficiency in G4-DNA better than in double-stranded DNA?

    OpenAIRE

    Woiczikowski, P. Benjamin; Kubař, Tomáš; Gutiérrez, Rafael; Cuniberti, Gianaurelio; Elstner, Marcus

    2010-01-01

    The electrical conduction properties of G4-DNA are investigated using a hybrid approach, which combines electronic structure calculations, molecular dynamics (MD) simulations, and the formulation of an effective tight-binding model Hamiltonian. Charge transport is studied by computing transmission functions along the MD trajectories. Though G4-DNA is structurally more stable than double-stranded DNA (dsDNA), our results strongly suggest that the potential improvement of the electrical transpo...

  19. Mechanochemical regulations of RPA's binding to ssDNA

    Science.gov (United States)

    Chen, Jin; Le, Shimin; Basu, Anindita; Chazin, Walter J.; Yan, Jie

    2015-03-01

    Replication protein A (RPA) is a ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein that serves to protect ssDNA from degradation and annealing, and as a template for recruitment of many downstream factors in virtually all DNA transactions in cell. During many of these transactions, DNA is tethered and is likely subject to force. Previous studies of RPA's binding behavior on ssDNA were conducted in the absence of force; therefore the RPA-ssDNA conformations regulated by force remain unclear. Here, using a combination of atomic force microscopy imaging and mechanical manipulation of single ssDNA tethers, we show that force mediates a switch of the RPA bound ssDNA from amorphous aggregation to a much more regular extended conformation. Further, we found an interesting non-monotonic dependence of the binding affinity on monovalent salt concentration in the presence of force. In addition, we discovered that zinc in micromolar concentrations drives ssDNA to a unique, highly stiff and more compact state. These results provide new mechanochemical insights into the influences and the mechanisms of action of RPA on large single ssDNA.

  20. Liquid-based cytology combined HPV-DNA genotyping in cervical cancer screening%液基细胞学检查联合人乳头瘤病毒基因分型检测在宫颈癌筛查中的应用

    Institute of Scientific and Technical Information of China (English)

    刘汉忠; 杨继洲; 肖兰; 卢叶; 戴一菲; 王岚

    2013-01-01

    目的 探讨液基细胞学检查(TCT)联合人乳头瘤病毒基因(HPV-DNA)分型检测在宫颈癌筛查中的应用价值.方法 对28 524例女性患者进行TCT检测,其中5 932例TCT诊断为不能明确意义的非典型鳞状细胞(ASCUS)及以上的患者均行HPV-DNA分型检测及宫颈活检,分析TCT和HPV感染与宫颈癌及癌前病变的相关性.结果 共检测样本28 524例,其中良性病变22 592例(79.20%),ASCUS 4 824例(16.91%),鳞状上皮内低度病变(LSIL) 776例(2.72%),鳞状上皮内高度病变(HSIL) 316例(1.11%),鳞状细胞癌(SCC) 16例(0.06%).HSIL符合率为97.47%(308/316例),SCC符合率为100%(16/16例).HPV-DNA分型检测5 932例,其中阳性1 486例,阳性率为25.05%.TCT诊断与活检结果比较,良性病变与CINⅠ、CINⅡ、CINⅢ比较差异有统计学意义(P<0.01); CINⅠ、CINⅡ、CINⅢ、SCC间比较差异有统计学意义(P<0.01).宫颈活检结果与HPV感染在炎症及CINⅠ、CINⅡ、CINⅢ、SCC组间比较差异有统计学意义(P<0.01).结论 TCT与活检,特别是针对高级病变及癌,符合率较高,特异性较强.TCT与HPV-DNA分型检测对诊断宫颈癌及癌前病变具有敏感、特异和早于形态学改变的特点,具有较高的临床应用价值和推广前景.%Objective To investigate the value of the liquid-based cytology (TCT) joint HPV-DNA genotyping in cervical cancer screening.Methods Liquid-based cytology (TCT) combined HPV-DNA genotyping,and TCT were detected in 28 524 women,including 5 932 of TCT cytological diagnosis of ASCUS or more patients underwent genotyping of HPV-DNA detection and cervical biopsy,TCT and HPV infection and cervical cancer and precancerous lesions to cervical biopsy results confirmed standard.Results The total number of samples tested were 28 524 cases.Benign reactive changes were 22 592 cases (79.20%),ASCUS 4 824 cases (16.91%),LSIL 776 cases (2.72 %),HSIL 316 cases (1.11%),and the SCC 16 cases (0.06 %),in which

  1. Natural transformation of bacteria by fragmented, damaged and ancient DNA

    DEFF Research Database (Denmark)

    Overballe-Petersen, Søren

    with fullgenome comparisons that the process has general relevance in extant bacteria. Our findings reveal that the large environmental reservoir of short and damaged DNA retains capacity for natural transformation, even after thousands of years. This describes for the first time a process by which cells can...... acquire functional genetic signatures of the deeper past. Moreover, not only can old DNA revert microbes to past genotypes, but damaged DNA can also produce new variants of already functional sequences. Besides, DNA fragments carry potential to combine functional domains in new ways. The identified novel...

  2. PNA Directed Sequence Addressed Self-Assembly of DNA Nanostructures

    DEFF Research Database (Denmark)

    Nielsen, Peter E.

    2008-01-01

    Peptide nucleic acids (PNA) can be designed to target duplex DNA with very high sequence specificity and efficiency via various binding modes. We have designed three domain PNA clamps, that bind stably to predefined decameric homopurine targets in large dsDNA mols. and via a third PNA domain...... sequence specifically recognize another PNA oligomer. We describe how such three domain PNAs have utility for assembling dsDNA grid and clover leaf structures, and in combination with SNAP-tag technol. of protein dsDNA structures. (c) 2008 American Institute of Physics. [on SciFinder (R)] Udgivelsesdato...

  3. PNA Directed Sequence Addressed Self-Assembly of DNA Nanostructures

    Science.gov (United States)

    Nielsen, Peter E.

    2008-10-01

    Peptide nucleic acids (PNA) can be designed to target duplex DNA with very high sequence specificity and efficiency via various binding modes. We have designed three domain PNA clamps, that bind stably to predefined decameric homopurine targets in large dsDNA molecules and via a third PNA domain sequence specifically recognize another PNA oligomer. We describe how such three domain PNAs have utility for assembling dsDNA grid and clover leaf structures, and in combination with SNAP-tag technology of protein dsDNA structures.

  4. Mitochondrial DNA, restoring Beethovens music.

    Science.gov (United States)

    Merheb, Maxime; Vaiedelich, Stéphane; Maniguet, Thiérry; Hänni, Catherine

    2016-01-01

    Great ancient composers have endured many obstacles and constraints which are very difficult to understand unless we perform the restoration process of ancient music. Species identification in leather used during manufacturing is the key step to start such a restoration process in order to produce a facsimile of a museum piano. Our study reveals the species identification in the leather covering the hammer head in a piano created by Erard in 1802. This is the last existing piano similar to the piano that Beethoven used with its leather preserved in its original state. The leather sample was not present in a homogeneous piece, yet combined with glue. Using a DNA extraction method that avoids PCR inhibitors; we discovered that sheep and cattle are the origin of the combination. To identify the species in the leather, we focused on the amounts of mitochondrial DNA in both leather and glue and results have led us to the conclusion that the leather used to cover the hammer head in this piano was made of cattle hide.

  5. DNA barcoding, phylogenetic relationships and speciation of snappers (genus Lutjanus)

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The phylogenetic relationships of 13 snapper species from the South China Sea have been established using the combined DNA sequences of three full-length mitochondrial genes (COI, COII and CYTB) and two partial nuclear genes (RAG1, RAG2). The 13 species (genus Lutjanus) were selected after DNA barcoding 72 individuals, representing 20 species. Our study suggests that although DNA barcoding aims to develop species identification systems, it may also be useful in the construction of phylogenies by aiding the selection of taxa. Combined mitochondrial and nuclear gene data has an advantage over an individual dataset because of its higher resolving power.

  6. Hierarchical-Multiplex DNA Patterns Mediated by Polymer Brush Nanocone Arrays That Possess Potential Application for Specific DNA Sensing.

    Science.gov (United States)

    Liu, Wendong; Liu, Xueyao; Ge, Peng; Fang, Liping; Xiang, Siyuan; Zhao, Xiaohuan; Shen, Huaizhong; Yang, Bai

    2015-11-11

    This paper provides a facile and cost-efficient method to prepare single-strand DNA (ssDNA) nanocone arrays and hierarchical DNA patterns that were mediated by poly(2-hydroxyethyl methacrylate) (PHEMA) brush. The PHEMA brush nanocone arrays with different morphology and period were fabricated via colloidal lithography. The hierarchical structure was prepared through the combination of colloidal lithography and traditional photolithography. The DNA patterns were easily achieved via grafting the amino group modified ssDNA onto the side chain of polymer brush, and the anchored DNA maintained their reactivity. The as-prepared ssDNA nanocone arrays can be applied for target DNA sensing with the detection limit reaching 1.65 nM. Besides, with the help of introducing microfluidic ideology, the hierarchical-multiplex DNA patterns on the same substrate could be easily achieved with each kind of pattern possessing one kind of ssDNA, which are promising surfaces for the preparation of rapid, visible, and multiplex DNA sensors.

  7. Tight-binding parameters for charge transfer along DNA

    CERN Document Server

    Hawke, L G D; Simserides, C

    2009-01-01

    We systematically examine all the tight-binding parameters pertinent to charge transfer along DNA. The $\\pi$ molecular structure of the four DNA bases (adenine, thymine, cytosine, and guanine) is investigated by using the linear combination of atomic orbitals method with a recently introduced parametrization. The HOMO and LUMO wavefunctions and energies of DNA bases are discussed and then used for calculating the corresponding wavefunctions of the two B-DNA base-pairs (adenine-thymine and guanine-cytosine). The obtained HOMO and LUMO energies of the bases are in good agreement with available experimental values. Our results are then used for estimating the complete set of charge transfer parameters between neighboring bases and also between successive base-pairs, considering all possible combinations between them, for both electrons and holes. The calculated microscopic quantities can be used in mesoscopic theoretical models of electron or hole transfer along the DNA double helix, as they provide the necessar...

  8. DNA-cell conjugates

    Energy Technology Data Exchange (ETDEWEB)

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2016-05-03

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  9. Characterization of muntjac DNA

    International Nuclear Information System (INIS)

    Sister chromatid exchange (SCE) in muntjac chromosomes is generally proportional to the chromosomal DNA content, but the SCE frequency is reduced in the heterochromatic neck region of the X chromosome. The physical properties of muntjac DNA and the kinetics of repair of UV damage in muntjac heterochromatin and euchromatin were examined and compared with the distribution of sister chromatid exchange

  10. Routine DNA testing

    Science.gov (United States)

    Routine DNA testing. It’s done once you’ve Marker-Assisted Breeding Pipelined promising Qantitative Trait Loci within your own breeding program and thereby established the performance-predictive power of each DNA test for your germplasm under your conditions. By then you are ready to screen your par...

  11. DNA sequences encoding erythropoietin

    Energy Technology Data Exchange (ETDEWEB)

    Lin, F.K.

    1987-10-27

    A purified and isolated DNA sequence is described consisting essentially of a DNA sequence encoding a polypeptide having an amino acid sequence sufficiently duplicative of that of erythropoietin to allow possession of the biological property of causing bone marrow cells to increase production of reticulocytes and red blood cells, and to increase hemoglobin synthesis or iron uptake.

  12. Recombinant DNA for Teachers.

    Science.gov (United States)

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  13. Protein–DNA Interactions

    NARCIS (Netherlands)

    Kovacic, L.; Boelens, R.

    2012-01-01

    The recognition of specific DNA sequences by proteins and the coupling to signaling events are fundamental occurrences that lie at the root of many cellular processes. Many examples of tight control by protein–DNA interactions can be found in such dynamic processes as transcription, replication and

  14. Characterization of muntjac DNA

    Energy Technology Data Exchange (ETDEWEB)

    Davis, R.C.

    1981-05-27

    Sister chromatid exchange (SCE) in muntjac chromosomes is generally proportional to the chromosomal DNA content, but the SCE frequency is reduced in the heterochromatic neck region of the X chromosome. The physical properties of muntjac DNA and the kinetics of repair of UV damage in muntjac heterochromatin and euchromatin were examined and compared with the distribution of sister chromatid exchange.

  15. Workshop on DNA repair.

    NARCIS (Netherlands)

    A.R. Lehmann (Alan); J.H.J. Hoeijmakers (Jan); A.A. van Zeeland (Albert); C.M.P. Backendorf (Claude); B.A. Bridges; A. Collins; R.P.D. Fuchs; G.P. Margison; R. Montesano; E. Moustacchi; A.T. Natarajan; M. Radman; A. Sarasin; E. Seeberg; C.A. Smith; M. Stefanini (Miria); L.H. Thompson; G.P. van der Schans; C.A. Weber (Christine); M.Z. Zdzienika

    1992-01-01

    textabstractA workshop on DNA repair with emphasis on eukaryotic systems was held, under the auspices of the EC Concerted Action on DNA Repair and Cancer, at Noordwijkerhout (The Netherlands) 14-19 April 1991. The local organization of the meeting was done under the auspices of the Medical Genetic C

  16. Extended DNA Tile Actuators

    DEFF Research Database (Denmark)

    Kristiansen, Martin; Kryger, Mille; Zhang, Zhao;

    2012-01-01

    A dynamic linear DNA tile actuator is expanded to three new structures of higher complexity. The original DNA actuator was constructed from a central roller strand which hybridizes with two piston strands by forming two half-crossover junctions. A linear expansion of the actuator is obtained...

  17. Clinical value of TCT combined with HPV-DNA detection in cervical cancer screening%液基薄层细胞学联合 HPV-DNA检测对宫颈癌患者筛查的临床价值

    Institute of Scientific and Technical Information of China (English)

    李媛媛

    2016-01-01

    Objective:To explore clinical value ThinPrep cytology test ( TCT) and HPV detection in cervical cancer screen-ing. Methods:The clinical data of the patients in the screening of cervical precancerous lesions were collected. Each patient was given TCT combined with HPV detection. Finally, after colposcopy cervical biopsy, the sensitivity, accuracy and false positive rate of single TCT, single HPV and TCT combined with HPV in the diagnosis of chronic cervical inflammation, cervical precancerous lesions and cervical cancer were compared. Results:The cervical cancer and precancerous lesions screening under colposcopy biopsy was done in 360 patients. Among the 3 screening methods, the combined application of TCT and HPV had the highest sensitivity and specificity, and could be used for diagnosing severe atypical hyperplasia ( CIN-III) and cervical carcinoma without missed diagnosis;however, it might miss some moderate dysplasia ( CIN-II) and mild atypical hyperplasia ( CIN-I) patients. Conclusions:The TCT combined with HPV-DNA detection can be used actively in the cervical precancerous lesions screening, and improve the accuracy of clinical diagno-sis. Moreover, combined with colposcopy biopsy of cervical cancer and precancerous lesions, it can provide a diagnostic basis for the clinical treatment, and is worthy of clinical application.%目的::探讨液基薄层细胞学与HPV检测联合应用后在宫颈癌患者筛查中的临床应用价值。方法:分析行宫颈癌前病变筛查的患者共360例临床资料,每位患者给予薄层液基细胞学联合HPV检测,最后经阴道镜下宫颈活检,比较单独TCT检查、单独HPV检查及两种检查联合检测在诊断患者宫颈慢性炎症、宫颈癌前病变及宫颈癌的敏感程度、准确性和假阳性率。结果:宫颈癌及癌前病变筛查患者,行阴道镜下活检的患者360例。3种筛选方法中,联合应用TCT及HPV的敏感性、特异性最

  18. Trans-dichlorooxovandium (IV) complex as a novel photoinducible DNA interstrand crosslinker for cancer therapy

    DEFF Research Database (Denmark)

    Somyajit, Kumar; Banik, Bhabatosh; Saxena, Sneha;

    2016-01-01

    -dichlorooxovanadium (IV) complex of pyrenyl terpyridine (VDC) is a novel photoinducible DNA crosslinking agent. By a combination of in vitro and ex vivo experiments including plasmid-based assays, we find that VDC forms monoadducts on the DNA and can be activated by UV-A and visible light to generate DNA interstrand...

  19. DNA extraction from Ascaris suum muscle tissue

    OpenAIRE

    Di Mito, Carmela; Betschart, Bruno

    2009-01-01

    A new method for the extraction of DNA from Ascaris suum muscle has been developed. It combines a standard SDS-based extraction with a plant DNA extraction procedure. The use of SDS and proteinase K allows the elimination of proteins, while CTAB and polyclar AT eliminate glycogen and polyphenols. The DNA thus obtained can easily be digested by endonucleases and amplified by PCR.

  20. The roles of adenoviral vectors and donor DNA structures on genome editing

    NARCIS (Netherlands)

    Holkers, Maarten

    2016-01-01

    Accurate and efficient genome editing is primarily dependent on the generation of a sequence-specific, genomic double-stranded DNA break (DSB) combined with the introduction of an exogenous DNA template into target cells. The exogenous template, called donor DNA, normally contains the foreign sequen

  1. Archaeal DNA replication.

    Science.gov (United States)

    Kelman, Lori M; Kelman, Zvi

    2014-01-01

    DNA replication is essential for all life forms. Although the process is fundamentally conserved in the three domains of life, bioinformatic, biochemical, structural, and genetic studies have demonstrated that the process and the proteins involved in archaeal DNA replication are more similar to those in eukaryal DNA replication than in bacterial DNA replication, but have some archaeal-specific features. The archaeal replication system, however, is not monolithic, and there are some differences in the replication process between different species. In this review, the current knowledge of the mechanisms governing DNA replication in Archaea is summarized. The general features of the replication process as well as some of the differences are discussed. PMID:25421597

  2. DNA repair protocols

    DEFF Research Database (Denmark)

    Bjergbæk, Lotte

    In its 3rd edition, this Methods in Molecular Biology(TM) book covers the eukaryotic response to genomic insult including advanced protocols and standard techniques in the field of DNA repair. Offers expert guidance for DNA repair, recombination, and replication. Current knowledge of the mechanisms...... that regulate DNA repair has grown significantly over the past years with technology advances such as RNA interference, advanced proteomics and microscopy as well as high throughput screens. The third edition of DNA Repair Protocols covers various aspects of the eukaryotic response to genomic insult including...... recent advanced protocols as well as standard techniques used in the field of DNA repair. Both mammalian and non-mammalian model organisms are covered in the book, and many of the techniques can be applied with only minor modifications to other systems than the one described. Written in the highly...

  3. DNA interaction with flavone and hydroxyflavones

    Science.gov (United States)

    Vitorino, Joana; Sottomayor, M. J.

    2010-06-01

    In recent years flavonoids have attracted a great interest as potential therapeutic drugs against a wide range of diseases. While the antioxidant activity of these natural polyphenolic compounds is well known, their binding to DNA characteristics is not fully understood despite the fact that many of them exert their biological effects by reversibly binding to nucleic acids. The present study aims to investigate the interaction of flavone and four hydroxyflavone isomers with double stranded DNA, occurring in bulk solution. A combination of micro-DSC and UV spectroscopy has been used to study the effect of these compounds and of their structure on the structure and stability of the DNA molecule. The characteristics of DNA thermal denaturation have been used as a measure of the effect of the compounds on the stability of the double helix. Micro-DSC has been used to determine the temperature dependence of the heat capacity of the process of thermal denaturation of DNA in solutions containing flavone and the following hydroxyflavones: 3-hydroxyflavone, 5-hydroxyflavone, 6-hydroxyflavone and 7-hydroxyflavone. The observed enthalpy of transition and the transition temperature of DNA thermal denaturation were determined for each compound. UV thermal denaturation curves were also recorded, under the same experimental conditions as the DSC measurements. The transition temperature values and the thermodynamic parameters of thermal denaturation were determined. Differential scanning calorimetry and ultraviolet spectroscopy measurements have evidenced an interaction between the studied flavones and DNA, showing a stabilization of DNA structure, due mainly to intercalation of the flavones. The results also show a noteworthy effect of the structure of the hydroxyflavone isomers on this stabilization.

  4. Diverse Applications of Environmental DNA Methods in Parasitology.

    Science.gov (United States)

    Bass, David; Stentiford, Grant D; Littlewood, D T J; Hartikainen, Hanna

    2015-10-01

    Nucleic acid extraction and sequencing of genes from organisms within environmental samples encompasses a variety of techniques collectively referred to as environmental DNA or 'eDNA'. The key advantages of eDNA analysis include the detection of cryptic or otherwise elusive organisms, large-scale sampling with fewer biases than specimen-based methods, and generation of data for molecular systematics. These are particularly relevant for parasitology because parasites can be difficult to locate and are morphologically intractable and genetically divergent. However, parasites have rarely been the focus of eDNA studies. Focusing on eukaryote parasites, we review the increasing diversity of the 'eDNA toolbox'. Combining eDNA methods with complementary tools offers much potential to understand parasite communities, disease risk, and parasite roles in broader ecosystem processes such as food web structuring and community assembly.

  5. Osmotic pressure: resisting or promoting DNA ejection from phage

    CERN Document Server

    Jeembaeva, Meerim; Larsson, Frida; Evilevitch, Alex

    2008-01-01

    Recent in vitro experiments have shown that DNA ejection from bacteriophage can be partially stopped by surrounding osmotic pressure when ejected DNA is digested by DNase I on the course of ejection. We argue in this work by combination of experimental techniques (osmotic suppression without DNaseI monitored by UV absorbance, pulse-field electrophoresis, and cryo-EM visualization) and simple scaling modeling that intact genome (i.e. undigested) ejection in a crowded environment is, on the contrary, enhanced or eventually complete with the help of a pulling force resulting from DNA condensation induced by the osmotic stress itself. This demonstrates that in vivo, the osmotically stressed cell cytoplasm will promote phage DNA ejection rather than resisting it. The further addition of DNA-binding proteins under crowding conditions is shown to enhance the extent of ejection. We also found some optimal crowding conditions for which DNA content remaining in the capsid upon ejection is maximum, which correlates well...

  6. ENA多肽抗体谱检测结合抗ds-DNA及补体C3测定在SLE诊断上的应用%Spectrum of ENA polypeptide antibodies combined with anti ds-DNA and C3 determination in the diagnosis of systemic lupus erythematosus

    Institute of Scientific and Technical Information of China (English)

    吕春兰; 都青; 杨荣生; 曹宪华

    2011-01-01

    in orther rheumatic diseases,the specificity is relatively high. Anti- SSA, anti- UIRNP in SLE was 36.8%, 27.6% , and orther rheumatic diseases are also detected,specificity is relatively low. In other rheumatic diseases the positive rate about anti SSA, anti SSB, anti UIRNP were 50 %, 43 %,25% respectively in SS;The positive rate about anti SSA.anti Jo I were 25%,25% respectively in PPS;The positive rates about anti SSA,anti UIRNP,anti-Jo-1, antiDM53 were 25%,25%,12. 5%,37. 5% respectively in DM;the positive rates about antiSSA,anti UIRNP,anti-- A/54 were 32.6% ,11.9% ,15.2%respectively in RF. Complement C3 in the determination of SLE values was low,ranging from 0.46 ± 0.12 , compared with orther rheumatic diseases, (P<0.01) significant differences. Conclusion The combination of anti- ENA antibodies, anti, ds- DNA antibodies and complement C3 determination in the diagnosis of SLE have significant clinical significance.

  7. Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

    Science.gov (United States)

    Schellenberg, Matthew J; Perera, Lalith; Strom, Christina N; Waters, Crystal A; Monian, Brinda; Appel, C Denise; Vilas, Caroline K; Williams, Jason G; Ramsden, Dale A; Williams, R Scott

    2016-05-01

    Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA-protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg(2+)-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg(2+) binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons. PMID:27060144

  8. DNA vaccines against influenza.

    Science.gov (United States)

    Stachyra, Anna; Góra-Sochacka, Anna; Sirko, Agnieszka

    2014-01-01

    Genetic vaccine technology has been considerably developed within the last two decades. This cost effective and promising strategy can be applied for therapy of cancers and for curing allergy, chronic and infectious diseases, such as a seasonal and pandemic influenza. Despite numerous advantages, several limitations of this technology reduce its performance and can retard its commercial exploitation in humans and its veterinary applications. Inefficient delivery of the DNA vaccine into cells of immunized individuals results in low intracellular supply of suitable expression cassettes encoding an antigen, in its low expression level and, in turn, in reduced immune responses against the antigen. Improvement of DNA delivery into the host cells might significantly increase effectiveness of the DNA vaccine. A vast array of innovative methods and various experimental strategies have been applied in order to enhance the effectiveness of DNA vaccines. They include various strategies improving DNA delivery as well as expression and immunogenic potential of the proteins encoded by the DNA vaccines. Researchers focusing on DNA vaccines against influenza have applied many of these strategies. Recent examples of the most successful modern approaches are discussed in this review.

  9. DNA mini-barcodes.

    Science.gov (United States)

    Hajibabaei, Mehrdad; McKenna, Charly

    2012-01-01

    Conventional DNA barcoding uses an approximately 650 bp DNA barcode of the mitochondrial gene COI for species identification in animal groups. Similar size fragments from chloroplast genes have been proposed as barcode markers for plants. While PCR amplification and sequencing of a 650 bp fragment is consistent in freshly collected and well-preserved specimens, it is difficult to obtain a full-length barcode in older museum specimens and samples which have been preserved in formalin or similar DNA-unfriendly preservatives. A comparable issue may prevent effective DNA-based authentication and testing in processed biological materials, such as food products, pharmaceuticals, and nutraceuticals. In these cases, shorter DNA sequences-mini-barcodes-have been robustly recovered and shown to be effective in identifying majority of specimens to a species level. Furthermore, short DNA regions can be utilized via high-throughput sequencing platforms providing an inexpensive and comprehensive means of large-scale species identification. These properties of mini-barcodes, coupled with the availability of standardized and universal primers make mini-barcodes a feasible option for DNA barcode analysis in museum samples and applied diagnostic and environmental biodiversity analysis.

  10. HIV DNA Integration

    Science.gov (United States)

    Craigie, Robert; Bushman, Frederic D.

    2012-01-01

    Retroviruses are distinguished from other viruses by two characteristic steps in the viral replication cycle. The first is reverse transcription, which results in the production of a double-stranded DNA copy of the viral RNA genome, and the second is integration, which results in covalent attachment of the DNA copy to host cell DNA. The initial catalytic steps of the integration reaction are performed by the virus-encoded integrase (IN) protein. The chemistry of the IN-mediated DNA breaking and joining steps is well worked out, and structures of IN-DNA complexes have now clarified how the overall complex assembles. Methods developed during these studies were adapted for identification of IN inhibitors, which received FDA approval for use in patients in 2007. At the chromosomal level, HIV integration is strongly favored in active transcription units, which may promote efficient viral gene expression after integration. HIV IN binds to the cellular factor LEDGF/p75, which promotes efficient infection and tethers IN to favored target sites. The HIV integration machinery must also interact with many additional host factors during infection, including nuclear trafficking and pore proteins during nuclear entry, histones during initial target capture, and DNA repair proteins during completion of the DNA joining steps. Models for some of the molecular mechanisms involved have been proposed, but important details remain to be clarified. PMID:22762018

  11. Quantitive DNA Fiber Mapping

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Chun-Mei; Wang, Mei; Greulich-Bode, Karin M.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-01-28

    Several hybridization-based methods used to delineate single copy or repeated DNA sequences in larger genomic intervals take advantage of the increased resolution and sensitivity of free chromatin, i.e., chromatin released from interphase cell nuclei. Quantitative DNA fiber mapping (QDFM) differs from the majority of these methods in that it applies FISH to purified, clonal DNA molecules which have been bound with at least one end to a solid substrate. The DNA molecules are then stretched by the action of a receding meniscus at the water-air interface resulting in DNA molecules stretched homogeneously to about 2.3 kb/{micro}m. When non-isotopically, multicolor-labeled probes are hybridized to these stretched DNA fibers, their respective binding sites are visualized in the fluorescence microscope, their relative distance can be measured and converted into kilobase pairs (kb). The QDFM technique has found useful applications ranging from the detection and delineation of deletions or overlap between linked clones to the construction of high-resolution physical maps to studies of stalled DNA replication and transcription.

  12. What Controls DNA Looping?

    Directory of Open Access Journals (Sweden)

    Pamela J. Perez

    2014-08-01

    Full Text Available The looping of DNA provides a means of communication between sequentially distant genomic sites that operate in tandem to express, copy, and repair the information encoded in the DNA base sequence. The short loops implicated in the expression of bacterial genes suggest that molecular factors other than the naturally stiff double helix are involved in bringing the interacting sites into close spatial proximity. New computational techniques that take direct account of the three-dimensional structures and fluctuations of protein and DNA allow us to examine the likely means of enhancing such communication. Here, we describe the application of these approaches to the looping of a 92 base-pair DNA segment between the headpieces of the tetrameric Escherichia coli Lac repressor protein. The distortions of the double helix induced by a second protein—the nonspecific nucleoid protein HU—increase the computed likelihood of looping by several orders of magnitude over that of DNA alone. Large-scale deformations of the repressor, sequence-dependent features in the DNA loop, and deformability of the DNA operators also enhance looping, although to lesser degrees. The correspondence between the predicted looping propensities and the ease of looping derived from gene-expression and single-molecule measurements lends credence to the derived structural picture.

  13. DNA/MVA Vaccines for HIV/AIDS.

    Science.gov (United States)

    Iyer, Smita S; Amara, Rama R

    2014-01-01

    Since the initial proof-of-concept studies examining the ability of antigen-encoded plasmid DNA to serve as an immunogen, DNA vaccines have evolved as a clinically safe and effective platform for priming HIV-specific cellular and humoral responses in heterologous "prime-boost" vaccination regimens. Direct injection of plasmid DNA into the muscle induces T- and B-cell responses against foreign antigens. However, the insufficient magnitude of this response has led to the development of approaches for enhancing the immunogenicity of DNA vaccines. The last two decades have seen significant progress in the DNA-based vaccine platform with optimized plasmid constructs, improved delivery methods, such as electroporation, the use of molecular adjuvants and novel strategies combining DNA with viral vectors and subunit proteins. These innovations are paving the way for the clinical application of DNA-based HIV vaccines. Here, we review preclinical studies on the DNA-prime/modified vaccinia Ankara (MVA)-boost vaccine modality for HIV. There is a great deal of interest in enhancing the immunogenicity of DNA by engineering DNA vaccines to co-express immune modulatory adjuvants. Some of these adjuvants have demonstrated encouraging results in preclinical and clinical studies, and these data will be examined, as well.

  14. DNA/MVA Vaccines for HIV/AIDS

    Directory of Open Access Journals (Sweden)

    Smita S. Iyer

    2014-02-01

    Full Text Available Since the initial proof-of-concept studies examining the ability of antigen-encoded plasmid DNA to serve as an immunogen, DNA vaccines have evolved as a clinically safe and effective platform for priming HIV-specific cellular and humoral responses in heterologous “prime-boost” vaccination regimens. Direct injection of plasmid DNA into the muscle induces T- and B-cell responses against foreign antigens. However, the insufficient magnitude of this response has led to the development of approaches for enhancing the immunogenicity of DNA vaccines. The last two decades have seen significant progress in the DNA-based vaccine platform with optimized plasmid constructs, improved delivery methods, such as electroporation, the use of molecular adjuvants and novel strategies combining DNA with viral vectors and subunit proteins. These innovations are paving the way for the clinical application of DNA-based HIV vaccines. Here, we review preclinical studies on the DNA-prime/modified vaccinia Ankara (MVA-boost vaccine modality for HIV. There is a great deal of interest in enhancing the immunogenicity of DNA by engineering DNA vaccines to co-express immune modulatory adjuvants. Some of these adjuvants have demonstrated encouraging results in preclinical and clinical studies, and these data will be examined, as well.

  15. DNA-PKcs is critical for telomere capping

    Energy Technology Data Exchange (ETDEWEB)

    Gilley, David; Tanaka, Hiromi; Hande, M. Prakash; Kurimasa,Akihiro; Li, Gloria C.; Chen, David J.

    2001-04-10

    The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is critical for DNA repair via the non-homologous end joining (NHEJ) pathway. Previously, it was reported that bone marrow cells and spontaneously transformed fibroblasts from SCID (severe combined immunodeficiency) mice have defects in telomere maintenance. The genetically defective SCID mouse arose spontaneously from its parental strain CB17. One known genomic alteration in SCID mice is a truncation of the extreme carboxyl-terminus of DNA-PKcs, but other as yet unidentified alterations may also exist. We have used a defined system, the DNA-PKcs knockout mouse, to investigate specifically the role DNA-PKcs specifically plays in telomere maintenance. We report that primary mouse embryonic fibroblasts (MEFs) and primary cultured kidney cells from 6-8 month old DNA-PKcs deficient mice accumulate a large number of telomere fusions, yet still retain wildtype telomere length. Thus, the phenotype of this defect separates the two-telomere related phenotypes, capping and length maintenance. DNA-PKcs deficient MEFs also exhibit elevated levels of chromosome fragments and breaks, which correlate with increased telomere fusions. Based on the high levels of telomere fusions observed in DNA-PKcs deficient cells, we conclude that DNA-PKcs plays an important capping role at the mammalian telomere.

  16. Radiation-induced DNA damage and DNA repair

    International Nuclear Information System (INIS)

    Although DNA undergoes various types of damage from radiation, active oxygen, and the like, a living body has a plurality of DNA repair mechanisms responding to the types of DNA damage. On the other hand, there are a system that results in cell death if the repair is impossible and a mechanism to lead to concretization if further repair is not accurately made. This paper explains the following items as the basic researches on these types of DNA damage and the repair mechanisms: (1) biological effects of DNA damage, (2) effect of DNA damage on DNA synthesis, and (3) effects of DNA damage on cells. It also explains the effects of radiation on cells with a focus on specific mechanism for (1) DNA damage caused by direct action due to radiation and by indirect action due mainly to active oxygen, and (2) DNA repair mechanism that works on DNA double-strand break (DSB). (A.O.)

  17. Apoptosis and DNA Methylation

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Huan X.; Hackett, James A. [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Nestor, Colm [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Breakthrough Research Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Dunican, Donncha S.; Madej, Monika; Reddington, James P. [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Pennings, Sari [Queen' s Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ (United Kingdom); Harrison, David J. [Breakthrough Research Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Meehan, Richard R., E-mail: Richard.Meehan@hgu.mrc.ac.uk [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Breakthrough Research Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU (United Kingdom)

    2011-04-01

    Epigenetic mechanisms assist in maintaining gene expression patterns and cellular properties in developing and adult tissues. The molecular pathology of disease states frequently includes perturbation of DNA and histone methylation patterns, which can activate apoptotic pathways associated with maintenance of genome integrity. This perspective focuses on the pathways linking DNA methyltransferases and methyl-CpG binding proteins to apoptosis, and includes new bioinformatic analyses to characterize the evolutionary origin of two G/T mismatch-specific thymine DNA glycosylases, MBD4 and TDG.

  18. Apoptosis and DNA Methylation

    Directory of Open Access Journals (Sweden)

    Richard R. Meehan

    2011-04-01

    Full Text Available Epigenetic mechanisms assist in maintaining gene expression patterns and cellular properties in developing and adult tissues. The molecular pathology of disease states frequently includes perturbation of DNA and histone methylation patterns, which can activate apoptotic pathways associated with maintenance of genome integrity. This perspective focuses on the pathways linking DNA methyltransferases and methyl-CpG binding proteins to apoptosis, and includes new bioinformatic analyses to characterize the evolutionary origin of two G/T mismatch-specific thymine DNA glycosylases, MBD4 and TDG.

  19. DNA from keratinous tissue

    DEFF Research Database (Denmark)

    Olsen, Maia E.; Bengtsson, Camilla Friis; Bertelsen, Mads Frost;

    2012-01-01

    feathers. As with nail and hair, we demonstrate that although DNA can, in general, be recovered from all parts of the feather, the quality of such DNA varies. As such, although one can expect a priori that genetic analyses are possible on the feather, for PCR based analyses, it is extremely difficult...... to predict the size of amplicon that can be used in such analyses. However, PCR-free genetic analyses that can exploit much smaller DNA fragments may promise to be a powerful tool for future exploitation....

  20. "Artifactual" arsenate DNA

    DEFF Research Database (Denmark)

    Nielsen, Peter E

    2012-01-01

    The recent claim by Wolfe-Simon et al. that the Halomonas bacterial strain GFAJ-1 when grown in arsenate-containing medium with limiting phosphate is able to substitute phosphate with arsenate in biomolecules including nucleic acids and in particular DNA(1) arose much skepticism, primarily due...... to the very limited chemical stability of arsenate esters (see ref. 2 and references therein). A major part of the criticisms was concerned with the insufficient (bio)chemical evidence in the Wolfe-Simon study for the actual chemical incorporation of arsenate in DNA (and/or RNA). Redfield et al. now present...... evidence that the identification of arsenate DNA was artifactual....

  1. Antiparasitic DNA vaccines in 21st century.

    Science.gov (United States)

    Wedrychowicz, Halina

    2015-06-01

    Demands for effective vaccines to control parasitic diseases of humans and livestock have been recently exacerbated by the development of resistance of most pathogenic parasites to anti-parasitic drugs. Novel genomic and proteomic technologies have provided opportunities for the discovery and improvement of DNA vaccines which are relatively easy as well as cheap to fabricate and stable at room temperatures. However, their main limitation is rather poor immunogenicity, which makes it necessary to couple the antigens with adjuvant molecules. This paper review recent advances in the development of DNA vaccines to some pathogenic protozoa and helminths. Numerous studies were conducted over the past 14 years of 21st century, employing various administration techniques, adjuvants and new immunogenic antigens to increase efficacy of DNA vaccines. Unfortunately, the results have not been rewarding. Further research is necessary using more extensive combinations of antigens; alternate delivery systems and more efficient adjuvants based on knowledge of the immunomodulatory capacities of parasitic protozoa and helminths.

  2. Selective Trapping of DNA Using Glass Microcapillaries.

    Science.gov (United States)

    Rempfer, Georg; Ehrhardt, Sascha; Laohakunakorn, Nadanai; Davies, Gary B; Keyser, Ulrich F; Holm, Christian; de Graaf, Joost

    2016-08-23

    We show experimentally that an inexpensive glass microcapillary can accumulate λ-phage DNA at its tip and deliver the DNA into the capillary using a combination of electro-osmotic flow, pressure-driven flow, and electrophoresis. We develop an efficient simulation model based on the electrokinetic equations and the finite-element method to explain this phenomenon. As a proof of concept for the generality of this trapping mechanism we use our numerical model to explore the effect of the salt concentration, the capillary surface charge, the applied voltage, the pressure difference, and the mobility of the analyte molecules. Our results indicate that the simple microcapillary system has the potential to capture a wide range of analyte molecules based on their electrophoretic mobility that extends well beyond our experimental example of λ-phage DNA. Our method for separation and preconcentration of analytes therefore has implications for the development of low-cost lab-on-a-chip devices. PMID:27479470

  3. Lattice engineering through nanoparticle-DNA frameworks

    Science.gov (United States)

    Tian, Ye; Zhang, Yugang; Wang, Tong; Xin, Huolin L.; Li, Huilin; Gang, Oleg

    2016-06-01

    Advances in self-assembly over the past decade have demonstrated that nano- and microscale particles can be organized into a large diversity of ordered three-dimensional (3D) lattices. However, the ability to generate different desired lattice types from the same set of particles remains challenging. Here, we show that nanoparticles can be assembled into crystalline and open 3D frameworks by connecting them through designed DNA-based polyhedral frames. The geometrical shapes of the frames, combined with the DNA-assisted binding properties of their vertices, facilitate the well-defined topological connections between particles in accordance with frame geometry. With this strategy, different crystallographic lattices using the same particles can be assembled by introduction of the corresponding DNA polyhedral frames. This approach should facilitate the rational assembly of nanoscale lattices through the design of the unit cell.

  4. Potential predictive role of chemotherapy-induced changes of soluble CD40 ligand in untreated advanced pancreatic ductal adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Azzariti A

    2016-07-01

    Full Text Available Amalia Azzariti,1,* Oronzo Brunetti,2,* Letizia Porcelli,1 Giusi Graziano,3 Rosa Maria Iacobazzi,1 Michele Signorile,2 Aldo Scarpa,4 Vito Lorusso,2 Nicola Silvestris2 1Preclinical and Clinical Pharmacology Unit, 2Medical Oncology Unit, 3Scientific Direction, National Cancer Research Centre, Istituto Tumouri “Giovanni Paolo II”, Bari, 4ARC-NET Research Centre, University of Verona, Verona, Italy *These authors contributed equally to this work Abstract: Pancreas ductal adenocarcinoma lacks predictive biomarkers. CD40 is a member of the tumor necrosis factor superfamily. CD40–sCD40L interaction is considered to contribute to the promotion of tumor cell growth and angiogenesis. The aim of the present study was to investigate the role of serum sCD40L as a predictor in metastatic pancreatic cancer. We evaluated 27 consecutive pancreatic cancer patients treated with FOLFIRINOX (21 patients or gemcitabine plus nab-paclitaxel combination (six patients. The sCD40L level was measured in serum by enzyme-linked immunosorbent assay at baseline, at first evaluation (all patients, and at time to progression (18 patients. The radiological response was evaluated according to the Response Evaluation Criteria in Solid Tumors, Version 1.1. The Wilcoxon signed-rank test was used to compare pre–post treatment sCD40L levels with respect to clinical response, while Pearson’s correlation coefficient was used for the correlation between sCD40L and CA19.9 pre- and post-treatment. The Kruskal–Wallis test was also conducted for further comparisons. We observed a statistically significant reduction in the sCD40L level after 3 months of treatment in patients with partial response (11,718.05±7,097.13 pg/mL vs 4,689.42±5,409.96 pg/mL; P<0.01. Conversely, in patients with progressive disease, the biomarker statistically increased in the same time (9,351.51±7,356.91 pg/mL vs 22,282.92±11,629.35 pg/mL; P<0.01. This trend of sCD40L was confirmed in 18 patients

  5. gDNA-Prot: Predict DNA-binding proteins by employing support vector machine and a novel numerical characterization of protein sequence.

    Science.gov (United States)

    Zhang, Yan-Ping; Wuyunqiqige; Zheng, Wei; Liu, Shuyi; Zhao, Chunguang

    2016-10-01

    DNA-binding proteins are the functional proteins in cells, which play an important role in various essential biological activities. An effective and fast computational method gDNA-Prot is proposed to predict DNA-binding proteins in this paper, which is a DNA-binding predictor that combines the support vector machine classifier and a novel kind of feature called graphical representation. The DNA-binding protein sequence information was described with the 20 probabilities of amino acids and the 23 new numerical graphical representation features of a protein sequence, based on 23 physicochemical properties of 20 amino acids. The Principal Components Analysis (PCA) was employed as feature selection method for removing the irrelevant features and reducing redundant features. The Sigmod function and Min-max normalization methods for PCA were applied to accelerate the training speed and obtain higher accuracy. Experiments demonstrated that the Principal Components Analysis with Sigmod function generated the best performance. The gDNA-Prot method was also compared with the DNAbinder, iDNA-Prot and DNA-Prot. The results suggested that gDNA-Prot outperformed the DNAbinder and iDNA-Prot. Although the DNA-Prot outperformed gDNA-Prot, gDNA-Prot was faster and convenient to predict the DNA-binding proteins. Additionally, the proposed gNDA-Prot method is available at http://sourceforge.net/projects/gdnaprot.

  6. gDNA-Prot: Predict DNA-binding proteins by employing support vector machine and a novel numerical characterization of protein sequence.

    Science.gov (United States)

    Zhang, Yan-Ping; Wuyunqiqige; Zheng, Wei; Liu, Shuyi; Zhao, Chunguang

    2016-10-01

    DNA-binding proteins are the functional proteins in cells, which play an important role in various essential biological activities. An effective and fast computational method gDNA-Prot is proposed to predict DNA-binding proteins in this paper, which is a DNA-binding predictor that combines the support vector machine classifier and a novel kind of feature called graphical representation. The DNA-binding protein sequence information was described with the 20 probabilities of amino acids and the 23 new numerical graphical representation features of a protein sequence, based on 23 physicochemical properties of 20 amino acids. The Principal Components Analysis (PCA) was employed as feature selection method for removing the irrelevant features and reducing redundant features. The Sigmod function and Min-max normalization methods for PCA were applied to accelerate the training speed and obtain higher accuracy. Experiments demonstrated that the Principal Components Analysis with Sigmod function generated the best performance. The gDNA-Prot method was also compared with the DNAbinder, iDNA-Prot and DNA-Prot. The results suggested that gDNA-Prot outperformed the DNAbinder and iDNA-Prot. Although the DNA-Prot outperformed gDNA-Prot, gDNA-Prot was faster and convenient to predict the DNA-binding proteins. Additionally, the proposed gNDA-Prot method is available at http://sourceforge.net/projects/gdnaprot. PMID:27378005

  7. Synergy of Two Assembly Languages in DNA Nanostructures: Self-Assembly of Sequence-Defined Polymers on DNA Cages.

    Science.gov (United States)

    Chidchob, Pongphak; Edwardson, Thomas G W; Serpell, Christopher J; Sleiman, Hanadi F

    2016-04-01

    DNA base-pairing is the central interaction in DNA assembly. However, this simple four-letter (A-T and G-C) language makes it difficult to create complex structures without using a large number of DNA strands of different sequences. Inspired by protein folding, we introduce hydrophobic interactions to expand the assembly language of DNA nanotechnology. To achieve this, DNA cages of different geometries are combined with sequence-defined polymers containing long alkyl and oligoethylene glycol repeat units. Anisotropic decoration of hydrophobic polymers on one face of the cage leads to hydrophobically driven formation of quantized aggregates of DNA cages, where polymer length determines the cage aggregation number. Hydrophobic chains decorated on both faces of the cage can undergo an intrascaffold "handshake" to generate DNA-micelle cages, which have increased structural stability and assembly cooperativity, and can encapsulate small molecules. The polymer sequence order can control the interaction between hydrophobic blocks, leading to unprecedented "doughnut-shaped" DNA cage-ring structures. We thus demonstrate that new structural and functional modes in DNA nanostructures can emerge from the synergy of two interactions, providing an attractive approach to develop protein-inspired assembly modules in DNA nanotechnology. PMID:26998893

  8. Developing DNA nanotechnology using single-molecule fluorescence.

    Science.gov (United States)

    Tsukanov, Roman; Tomov, Toma E; Liber, Miran; Berger, Yaron; Nir, Eyal

    2014-06-17

    CONSPECTUS: An important effort in the DNA nanotechnology field is focused on the rational design and manufacture of molecular structures and dynamic devices made of DNA. As is the case for other technologies that deal with manipulation of matter, rational development requires high quality and informative feedback on the building blocks and final products. For DNA nanotechnology such feedback is typically provided by gel electrophoresis, atomic force microscopy (AFM), and transmission electron microscopy (TEM). These analytical tools provide excellent structural information; however, usually they do not provide high-resolution dynamic information. For the development of DNA-made dynamic devices such as machines, motors, robots, and computers this constitutes a major problem. Bulk-fluorescence techniques are capable of providing dynamic information, but because only ensemble averaged information is obtained, the technique may not adequately describe the dynamics in the context of complex DNA devices. The single-molecule fluorescence (SMF) technique offers a unique combination of capabilities that make it an excellent tool for guiding the development of DNA-made devices. The technique has been increasingly used in DNA nanotechnology, especially for the analysis of structure, dynamics, integrity, and operation of DNA-made devices; however, its capabilities are not yet sufficiently familiar to the community. The purpose of this Account is to demonstrate how different SMF tools can be utilized for the development of DNA devices and for structural dynamic investigation of biomolecules in general and DNA molecules in particular. Single-molecule diffusion-based Förster resonance energy transfer and alternating laser excitation (sm-FRET/ALEX) and immobilization-based total internal reflection fluorescence (TIRF) techniques are briefly described and demonstrated. To illustrate the many applications of SMF to DNA nanotechnology, examples of SMF studies of DNA hairpins and

  9. Extracellular DNA and F-actin as targets in antibiofilm cystic fibrosis therapy

    DEFF Research Database (Denmark)

    Tolker-Nielsen, Tim; Høiby, Niels

    2009-01-01

    -DNase combination treatment. The synergistic effect of the combined polyvalent anion-DNase treatment is most likely due to the capacity of polyvalent anions to dissociate DNA/F-actin bundles with bacteria and polysaccharides, leading to exposure of a greater number of DNase cleavage sites on the extracellular DNA...

  10. Experimental phase diagram of negatively supercoiled DNA measured by magnetic tweezers and fluorescence.

    Science.gov (United States)

    Vlijm, Rifka; Mashaghi, Alireza; Bernard, Stéphanie; Modesti, Mauro; Dekker, Cees

    2015-02-21

    The most common form of DNA is the well-known B-structure of double-helix DNA. Many processes in the cell, however, exert force and torque, inducing structural changes to the DNA that are vital to biological function. Virtually all DNA in cells is in a state of negative supercoiling, with a DNA structure that is complex. Using magnetic tweezers combined with fluorescence imaging, we here study DNA structure as a function of negative supercoiling at the single-molecule level. We classify DNA phases based on DNA length as a function of supercoiling, down to a very high negative supercoiling density σ of -2.5, and forces up to 4.5 pN. We characterize plectonemes using fluorescence imaging. DNA bubbles are visualized by the binding of fluorescently labelled RPA, a eukaryotic single-strand-binding protein. The presence of Z-DNA, a left-handed form of DNA, is probed by the binding of Zα77, the minimal binding domain of a Z-DNA-binding protein. Without supercoiling, DNA is in the relaxed B-form. Upon going toward negative supercoiling, plectonemic B-DNA is being formed below 0.6 pN. At higher forces and supercoiling densities down to about -1.9, a mixed state occurs with plectonemes, multiple bubbles and left-handed L-DNA. Around σ = -1.9, a buckling transition occurs after which the DNA end-to-end length linearly decreases when applying more negative turns, into a state that we interpret as plectonemic L-DNA. By measuring DNA length, Zα77 binding, plectoneme and ssDNA visualisation, we thus have mapped the co-existence of many DNA structures and experimentally determined the DNA phase diagram at (extreme) negative supercoiling.

  11. DNA Sampling Hook

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The DNA Sampling Hook is a significant improvement on a method of obtaining a tissue sample from a live fish in situ from an aquatic environment. A tissue sample...

  12. Whose DNA is this?

    DEFF Research Database (Denmark)

    Taroni, Franco; Biedermann, Alex; Vuille, Joëlle;

    2013-01-01

    evoked during the international conference "The hidden side of DNA profiles. Artifacts, errors and uncertain evidence" held in Rome (April 27th to 28th, 2012). Indeed, despite the fact that this conference brought together some of the world's leading forensic DNA specialists, it appeared clearly......This communication seeks to draw the attention of researchers and practitioners dealing with forensic DNA profiling analyses to the following question: is a scientist's report, offering support to a hypothesis according to which a particular individual is the source of DNA detected during...... the analysis of a stain, relevant from the point of view of a Court of Justice? This question relates to skeptical views previously voiced by commentators mainly in the judicial area, but is avoided by a large majority of forensic scientists. Notwithstanding, the pivotal role of this question has recently been...

  13. DNA sequencing conference, 2

    Energy Technology Data Exchange (ETDEWEB)

    Cook-Deegan, R.M. [Georgetown Univ., Kennedy Inst. of Ethics, Washington, DC (United States); Venter, J.C. [National Inst. of Neurological Disorders and Strokes, Bethesda, MD (United States); Gilbert, W. [Harvard Univ., Cambridge, MA (United States); Mulligan, J. [Stanford Univ., CA (United States); Mansfield, B.K. [Oak Ridge National Lab., TN (United States)

    1991-06-19

    This conference focused on DNA sequencing, genetic linkage mapping, physical mapping, informatics and bioethics. Several were used to study this sequencing and mapping. This article also discusses computer hardware and software aiding in the mapping of genes.

  14. DNA damage and carcinogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Stelow, R B

    1980-01-01

    Although cancer may arise as a result of many different types of molecular changes, there is little reason to doubt that changes to DNA are one of the more important ones in cancer initiation. Although DNA repair mechanisms seem able to eliminate a very large fraction of deleterious changes to DNA, we not only have little insight into the molecular mechanisms involved in such repair, but have a negligible amount of information to permit us to estimate the shape of dose response relations at low doses. The case of skin cancer is a special one, in that the average population is exposed to sufficient solar uv so that the effects of small increments in uv dose may be estimated. An approximate 85% reduction in DNA repair increases skin cancer incidence 10/sup 4/ fold.

  15. FBI's DNA analysis program

    Science.gov (United States)

    Brown, John R.

    1994-03-01

    Forensic DNA profiling technology is a significant law enforcement tool due to its superior discriminating power. Applying the principles of population genetics to the DNA profile obtained in violent crime investigations results in low frequency of occurrence estimates for the DNA profile. These estimates often range from a frequency of occurrence of 1 in 50 unrelated individuals to 1 in a million unrelated individuals or even smaller. It is this power to discriminate among individuals in the population that has propelled forensic DNA technology to the forefront of forensic testing in violent crime cases. Not only is the technology extremely powerful in including or excluding a criminal suspect as the perpetrator, but it also gives rise to the potential of identifying criminal suspects in cases where the investigators of unknown suspect cases have exhausted all other available leads.

  16. Kink solitons in DNA

    CERN Document Server

    Zdravković, S; Daniel, M

    2012-01-01

    We here examine the nonlinear dynamics of artificial homogeneous DNA chain relying on the plain-base rotator model. It is shown that such dynamics can exhibit kink and antikink solitons of sine-Gordon type. In that respect we propose possible experimental assays based on single molecule micromanipulation techniques. The aim of these experiments is to excite the rotational waves and to determine their speeds along excited DNA. We propose that these experiments should be conducted either for the case of double stranded (DS) or single stranded (SS) DNA. A key question is to compare the corresponding velocities of the rotational waves indicating which one is bigger. The ratio of these velocities appears to be related with the sign of the model parameter representing ratio of the hydrogen-bonding and the covalent-bonding interaction within the considered DNA chain.

  17. DNA fusion gene vaccines

    DEFF Research Database (Denmark)

    Holst, Peter Johannes; Bassi, Maria Rosaria; Thomsen, Allan Randrup;

    2010-01-01

    DNA vaccines are versatile and safe, but limited immunogenicity has prevented their use in the clinical setting. Experimentally, immunogenicity may be enhanced by the use of new delivery technologies, by coadministration of cytokines and pathogen-associated molecular patterns, or by fusion...... of antigens into molecular domains that enhance antigen presentation. More specifically, the immunogenicity of DNA vaccines may benefit from increased protein synthesis, increased T-cell help and MHC class I presentation, and the addition of a range of specific cytokines and pathogen-associated molecular...... with viral-vectored vaccines, various synergistic components may need to be incorporated into DNA vaccines. From the perspective of the future clinical use of DNA vaccines, it has been suggested that antigen presentation should be improved and cytokine coadministration attempted. However, even...

  18. DNA synthesis on discontinuous templates by human DNA polymerases: implications for non-homologous DNA recombination.

    OpenAIRE

    Islas, L; Fairley, C F; Morgan, W. F.

    1998-01-01

    DNA polymerases catalyze the synthesis of DNA using a continuous uninterrupted template strand. However, it has been shown that a 3'-->5' exonuclease-deficient form of the Klenow fragment of Escherichia coli DNA polymerase I as well as DNA polymerase of Thermus aquaticus can synthesize DNA across two unlinked DNA templates. In this study, we used an oligonucleotide-based assay to show that discontinuous DNA synthesis was present in HeLa cell extracts. DNA synthesis inhibitor studies as well a...

  19. Interaction of DNA and DNA-anti-DNA complexes to fibronectin

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, R.C.; Simpson, W.A.; Raghow, R.; Hasty, K.

    1986-03-01

    Fibronectin (Fn) is a large multidomain glycoprotein found in the basement membrane, on cell surface and in plasma. The interactions of Fn with DNA may be significant in glomerular deposition of DNA-anti-DNA complexes in patients with systemic lupus erythematosus (SLE). The authors examined the binding of DNA and DNA-anti-DNA complexes to Fn by a solid phase assay in which Fn was coated to microtiter plates and reacted with (/sup 3/H)DNA or DNA complexes with a monoclonal anti-DNA antibody. The optimal interaction of DNA with Fn occurs at <0.1M NaCl suggesting that the binding is charge dependent; the specificity of this binding was shown by competitive inhibition and locking experiments using anti-Fn. The binding was maximum at pH 6.5 and in the absence of Ca/sup 2 +/. The addition of Clq enhanced the binding of DNA and DNA-anti-DNA complexes to Fn, whereas heparan sulfate inhibited such binding. The monomeric or aggregated IgC did not bind Fn but aggregated IgG bound to Fn in the presence of Clq. Furthermore, DNA-anti-DNA complexes in sera from active SLE patients bound Fn which was enhanced in the presence of Clq; DNase abolished this binding indicating that the interaction of these complexes was mediated by DNA. These observations may partially explain the molecular mechanism(s) of the deposition of DNA-anti-DNA complexes in basement membrane.

  20. Patterning nanocrystals using DNA

    Energy Technology Data Exchange (ETDEWEB)

    Williams, Shara Carol

    2003-09-01

    One of the goals of nanotechnology is to enable programmed self-assembly of patterns made of various materials with nanometer-sized control. This dissertation describes the results of experiments templating arrangements of gold and semiconductor nanocrystals using 2'-deoxyribonucleic acid (DNA). Previously, simple DNA-templated linear arrangements of two and three nanocrystals structures have been made.[1] Here, we have sought to assemble larger and more complex nanostructures. Gold-DNA conjugates with 50 to 100 bases self-assembled into planned arrangements using strands of DNA containing complementary base sequences. We used two methods to increase the complexity of the arrangements: using branched synthetic doublers within the DNA covalent backbone to create discrete nanocrystal groupings, and incorporating the nanocrystals into a previously developed DNA lattice structure [2][3] that self-assembles from tiles made of DNA double-crossover molecules to create ordered nanoparticle arrays. In the first project, the introduction of a covalently-branched synthetic doubler reagent into the backbone of DNA strands created a branched DNA ''trimer.'' This DNA trimer templated various structures that contained groupings of three and four gold nanoparticles, giving promising, but inconclusive transmission electron microscopy (TEM) results. Due to the presence of a variety of possible structures in the reaction mixtures, and due to the difficulty of isolating the desired structures, the TEM and gel electrophoresis results for larger structures having four particles, and for structures containing both 5 and 10 nm gold nanoparticles were inconclusive. Better results may come from using optical detection methods, or from improved sample preparation. In the second project, we worked toward making two-dimensional ordered arrays of nanocrystals. We replicated and improved upon previous results for making DNA lattices, increasing the size of the lattices

  1. Expansion of the DNA Alphabet beyond Natural DNA Recognition.

    Science.gov (United States)

    Tateishi-Karimata, Hisae; Sugimoto, Naoki

    2016-07-15

    Simple and inexpensive DNA fibres: New, stable DNA structures are created by the binding of a small molecule to poly(A). Because these DNA fibres are formed from inexpensive materials by using very simple methods, DNA materials suitable for practical use such as information storage should be possible in the near future. PMID:27061868

  2. Ejer jeg mit DNA?

    DEFF Research Database (Denmark)

    Hoffmeyer, Jesper

    2010-01-01

    Havasupai-indianerne har så lidt som nogen andre mennesker selv frembragt deres DNA. Det repræsenterer en form for biologisk viden opsamlet gennem milliuoner af års forhistorie......Havasupai-indianerne har så lidt som nogen andre mennesker selv frembragt deres DNA. Det repræsenterer en form for biologisk viden opsamlet gennem milliuoner af års forhistorie...

  3. What Controls DNA Looping?

    OpenAIRE

    Perez, Pamela J.; Nicolas Clauvelin; Grosner, Michael A.; Colasanti, Andrew V.; Olson, Wilma K.

    2014-01-01

    The looping of DNA provides a means of communication between sequentially distant genomic sites that operate in tandem to express, copy, and repair the information encoded in the DNA base sequence. The short loops implicated in the expression of bacterial genes suggest that molecular factors other than the naturally stiff double helix are involved in bringing the interacting sites into close spatial proximity. New computational techniques that take direct account of the three-dimensional stru...

  4. DNA-Origami

    DEFF Research Database (Denmark)

    Voigt, Niels Vinther; Tørring, Thomas; Gothelf, Kurt Vesterager

    2010-01-01

    DNA-nanostrukturer giver nye muligheder for studier af individuelle molekyler. Ved at udnytte DNAs unikke selvsamlende egenskaber kan man designe systemer, hvorpå der kan studeres kemiske reaktioner, fluoroforer og biiomolekyler på enkeltmolekyle-niveau.......DNA-nanostrukturer giver nye muligheder for studier af individuelle molekyler. Ved at udnytte DNAs unikke selvsamlende egenskaber kan man designe systemer, hvorpå der kan studeres kemiske reaktioner, fluoroforer og biiomolekyler på enkeltmolekyle-niveau....

  5. Photofootprinting of DNA triplexes.

    OpenAIRE

    Lyamichev, V I; Voloshin, O N; Frank-Kamenetskii, M D; Soyfer, V N

    1991-01-01

    We have used a photofootprinting assay to study intermolecular and intramolecular DNA triplexes. The assay is based on the fact that the DNA duplex is protected against photodamage (specifically, against the formation of the (6-4) pyrimidine photoproducts) within a triplex structure. We have shown that this is the case for PyPuPu (YRR) as well as PyPuPy (YRY) triplexes. Using the photofootprinting assay, we have studied the triplex formation under a variety of experimentally defined condition...

  6. Introduction to DNA methods

    International Nuclear Information System (INIS)

    The purpose of this session is to discuss the various possibilities for detecting modifications in DNA after irradiation and whether these changes can be utilized as an indicator for the irradiation treatment of foods. The requirement to be fulfilled is that the method be able to distinguish irradiated food without the presence of a control sample, thus the measured response after irradiation must be large enough to supersede background levels from other treatments. Much work has been performed on the effects of radiation on DNA, particularly due to its importance in radiation biology. The main lesions of DNA as a result of irradiation are base damage, damage of the sugar moiety, single strand and double strand breaks. Crosslinking between bases also occurs, e.g. production of thymine dimers, or between DNA and protein. A valuable review on how to utilize these DNA changes for detection purposes has already appeared. Tables 1, 2 and 3 list the proposed methods of detecting changes in irradiated DNA, some identified products as examples for a possible irradiation indicator, in the case of immunoassay the substance used as antigen, and some selected literature references. In this short review, it is not intended to provide a complete literature survey

  7. DNA vaccines and intradermal vaccination by DNA tattooing.

    Science.gov (United States)

    Oosterhuis, K; van den Berg, J H; Schumacher, T N; Haanen, J B A G

    2012-01-01

    Over the past two decades, DNA vaccination has been developed as a method for the induction of immune responses. However, in spite of high expectations based on their efficacy in preclinical models, immunogenicity of first generation DNA vaccines in clinical trials was shown to be poor, and no DNA vaccines have yet been licensed for human use. In recent years significant progress has been made in the development of second generation DNA vaccines and DNA vaccine delivery methods. Here we review the key characteristics of DNA vaccines as compared to other vaccine platforms, and recent insights into the prerequisites for induction of immune responses by DNA vaccines will be discussed. We illustrate the development of second generation DNA vaccines with the description of DNA tattooing as a novel DNA delivery method. This technique has shown great promise both in a small animal model and in non-human primates and is currently under clinical evaluation.

  8. A new structural framework for integrating replication protein A into DNA processing machinery

    Energy Technology Data Exchange (ETDEWEB)

    Brosey, Chris; Yan, Chunli; Tsutakawa, Susan; Heller, William; Rambo, Robert; Tainer, John; Ivanov, Ivaylo; Chazin, Walter

    2013-01-17

    By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA's DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA's DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamic on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways.

  9. Radiation-induced damage to DNA

    International Nuclear Information System (INIS)

    This short survey focuses on the main radiation-induced base lesions that have been identified within cellular DNA. For this purpose, sensitive assays that are aimed at measuring a few modifications per 107 normal bases were set-up. In that respect high performance liquid chromatography - tandem mass spectrometry (CLHP-MS/MS) was found to be able to single out the formation of 9 oxidized nucleosides and two modified nucleo-bases out of the 70 oxidative base lesions that have been identified in model systems. As a striking result, it was found that in the DNA of γ-irradiated human monocytes, the formamide-pyrimidine derivative of guanine is produced in a higher yield than the ubiquitous 8-oxo-7,8-dihydro-guanine damage, both arising from the same radical precursor. However, relatively high doses of ionizing radiation (> 20 Gy) have to be applied in order to detect an increase in the level of the damage. This is due to the low efficiency for both low and high LET radiations to generate oxidative damage to DNA on one hand and the occurrence of artifactual oxidation of the overwhelming normal bases during DNA extraction on the other hand. Interestingly, a modified comet assays that involves the combined use of the alkaline single gel electrophoretic technique and DNA repair N-glycosylases has allowed the detection of three main types of radiation-induced damage within the dose range 0.3 Gy -10 Gy. It appears that the total of frank DNA strand breaks and alkali-labile sites is similar to the sum of oxidized pyrimidine bases and modified purine bases that are recognized by the endonuclease Ill protein and the formamide-pyrimidine DNA N-glycosylase respectively. (author)

  10. Thrombelastography and biomarker profiles in acute coagulopathy of trauma: a prospective study

    Directory of Open Access Journals (Sweden)

    Larsen Claus F

    2011-10-01

    Full Text Available Abstract Background Severe injury induces an acute coagulopathy associated with increased mortality. This study compared the Thrombelastography (TEG and biomarker profiles upon admission in trauma patients. Methods Prospective observational study of 80 trauma patients admitted to a Level I Trauma Centre. Data on demography, biochemistry including standard coagulation tests, hematology, transfusions, Injury Severity Score (ISS and TEG were recorded. Retrospective analysis of thawed plasma/serum for biomarkers reflecting tissue injury (histone-complexed DNA fragments, sympathoadrenal activation (adrenaline, noradrenaline, coagulation activation/inhibition and fibrinolysis (sCD40L, protein C, activated Protein C, tissue-type plasminogen activator, plasminogen activator inhibitor-1, D-dimer, prothrombinfragment 1+2, plasmin/α2-antiplasmin complex, thrombin/antithrombin complex, tissue factor pathway inhibitor, antithrombin, von willebrand factor, factor XIII. Comparison of patients stratified according to ISS/TEG maximum clot strength. Linear regression analysis of variables associated with clot strength. Results Trauma patients had normal (86%, hypercoagulable (11% or hypocoagulable (1% TEG clot strength; one had primary hyperfibrinolysis. Hypercoagulable patients had higher age, fibrinogen and platelet count (all p 10 red blood cells the initial 24 h. Patients with normal or hypercoagulable TEG clot strength had comparable biomarker profiles, but the few patients with hypocoagulable TEG clot strength and/or hyperfibrinolysis had very different biomarker profiles. Increasing ISS was associated with higher levels of catecholamines, histone-complexed DNA fragments, sCD40L, activated protein C and D-dimer and reduced levels of non-activated protein C, antithrombin, fibrinogen and factor XIII (all p 26. In patients with ISS > 26, adrenaline and sCD40L were independently negatively associated with clot strength. Conclusions Trauma patients displayed

  11. Computational Design of DNA-Binding Proteins.

    Science.gov (United States)

    Thyme, Summer; Song, Yifan

    2016-01-01

    Predicting the outcome of engineered and naturally occurring sequence perturbations to protein-DNA interfaces requires accurate computational modeling technologies. It has been well established that computational design to accommodate small numbers of DNA target site substitutions is possible. This chapter details the basic method of design used in the Rosetta macromolecular modeling program that has been successfully used to modulate the specificity of DNA-binding proteins. More recently, combining computational design and directed evolution has become a common approach for increasing the success rate of protein engineering projects. The power of such high-throughput screening depends on computational methods producing multiple potential solutions. Therefore, this chapter describes several protocols for increasing the diversity of designed output. Lastly, we describe an approach for building comparative models of protein-DNA complexes in order to utilize information from homologous sequences. These models can be used to explore how nature modulates specificity of protein-DNA interfaces and potentially can even be used as starting templates for further engineering. PMID:27094297

  12. Estimating the entropy of DNA sequences.

    Science.gov (United States)

    Schmitt, A O; Herzel, H

    1997-10-01

    The Shannon entropy is a standard measure for the order state of symbol sequences, such as, for example, DNA sequences. In order to incorporate correlations between symbols, the entropy of n-mers (consecutive strands of n symbols) has to be determined. Here, an assay is presented to estimate such higher order entropies (block entropies) for DNA sequences when the actual number of observations is small compared with the number of possible outcomes. The n-mer probability distribution underlying the dynamical process is reconstructed using elementary statistical principles: The theorem of asymptotic equi-distribution and the Maximum Entropy Principle. Constraints are set to force the constructed distributions to adopt features which are characteristic for the real probability distribution. From the many solutions compatible with these constraints the one with the highest entropy is the most likely one according to the Maximum Entropy Principle. An algorithm performing this procedure is expounded. It is tested by applying it to various DNA model sequences whose exact entropies are known. Finally, results for a real DNA sequence, the complete genome of the Epstein Barr virus, are presented and compared with those of other information carriers (texts, computer source code, music). It seems as if DNA sequences possess much more freedom in the combination of the symbols of their alphabet than written language or computer source codes. PMID:9344742

  13. The colibactin warhead crosslinks DNA

    Science.gov (United States)

    Vizcaino, Maria I.; Crawford, Jason M.

    2015-05-01

    Members of the human microbiota are increasingly being correlated to human health and disease states, but the majority of the underlying microbial metabolites that regulate host-microbe interactions remain largely unexplored. Select strains of Escherichia coli present in the human colon have been linked to the initiation of inflammation-induced colorectal cancer through an unknown small-molecule-mediated process. The responsible non-ribosomal peptide-polyketide hybrid pathway encodes ‘colibactin’, which belongs to a largely uncharacterized family of small molecules. Genotoxic small molecules from this pathway that are capable of initiating cancer formation have remained elusive due to their high instability. Guided by metabolomic analyses, here we employ a combination of NMR spectroscopy and bioinformatics-guided isotopic labelling studies to characterize the colibactin warhead, an unprecedented substituted spirobicyclic structure. The warhead crosslinks duplex DNA in vitro, providing direct experimental evidence for colibactin's DNA-damaging activity. The data support unexpected models for both colibactin biosynthesis and its mode of action.

  14. Origin DNA Melting and Unwinding in DNA Replication

    OpenAIRE

    Gai, Dahai; Chang, Y Paul; Chen, Xiaojiang S.

    2010-01-01

    Genomic DNA replication is a necessary step in the life cycles of all organisms. To initiate DNA replication, the double-stranded DNA (dsDNA) at the origin of replication must be separated or melted; this melted region is propagated and a mature replication fork is formed. To accomplish origin recognition, initial DNA melting, and the eventual formation of a replication fork, coordinated activity of initiators, helicases, and other cellular factors are required. In this review, we focus on re...

  15. DNA display I. Sequence-encoded routing of DNA populations.

    OpenAIRE

    Halpin, David R; Pehr B Harbury

    2004-01-01

    Recently reported technologies for DNA-directed organic synthesis and for DNA computing rely on routing DNA populations through complex networks. The reduction of these ideas to practice has been limited by a lack of practical experimental tools. Here we describe a modular design for DNA routing genes, and routing machinery made from oligonucleotides and commercially available chromatography resins. The routing machinery partitions nanomole quantities of DNA into physically distinct subpools ...

  16. Hydrocodone Combination Products

    Science.gov (United States)

    Codiclear DH® (as a combination product containing Guaifenesin, Hydrocodone) ... EndaCof XP® (as a combination product containing Guaifenesin, Hydrocodone) ... Entuss® (as a combination product containing Guaifenesin, Hydrocodone)

  17. DNA Barcoding for Honey Biodiversity

    Directory of Open Access Journals (Sweden)

    Alice Valentini

    2010-04-01

    Full Text Available Honey is produced by honeybees from nectar and from secretions of living plants. It reflects the honeybees’ diet and the local plant communities. Honey also shows different plant compositions in different geographical locations. We propose a new method for studying the plant diversity and the geographical origin of honey using a DNA barcoding approach that combines universal primers and massive parallel pyrosequencing. To test this method we use two commercial honeys, one from a regional origin and one composed of a worldwide mix of different honeys. We demonstrate that the method proposed here is fast, simple to implement, more robust than classical methods, and therefore suitable for analyzing plant diversity in honey.

  18. Defects of mitochondrial DNA replication.

    Science.gov (United States)

    Copeland, William C

    2014-09-01

    Mitochondrial DNA is replicated by DNA polymerase γ in concert with accessory proteins such as the mitochondrial DNA helicase, single-stranded DNA binding protein, topoisomerase, and initiating factors. Defects in mitochondrial DNA replication or nucleotide metabolism can cause mitochondrial genetic diseases due to mitochondrial DNA deletions, point mutations, or depletion, which ultimately cause loss of oxidative phosphorylation. These genetic diseases include mitochondrial DNA depletion syndromes such as Alpers or early infantile hepatocerebral syndromes, and mitochondrial DNA deletion disorders, such as progressive external ophthalmoplegia, ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy. This review focuses on our current knowledge of genetic defects of mitochondrial DNA replication (POLG, POLG2, C10orf2, and MGME1) that cause instability of mitochondrial DNA and mitochondrial disease.

  19. Pitfalls in the analysis of ancient human mtDNA

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The retrieval of DNA from ancient human specimens is not always successful owing to DNA deterioration and contamination although it is vital to provide new insights into the genetic structure of ancient people and to reconstruct the past history. Normally, only short DNA fragments can be retrieved from the ancient specimens. How to identify the authenticity of DNA obtained and to uncover the information it contained are difficult. We employed the ancient mtDNAs reported from Central Asia (including Xinjiang, China) as an example to discern potentially extraneous DNA contamination based on the updated mtDNA phylogeny derived from mtDNA control region, coding region, as well as complete sequence information. Our results demonstrated that many mtDNAs reported are more or less problematic. Starting from a reliable mtDNA phylogeney and combining the available modern data into analysis, one can ascertain the authenticity of the ancient DNA, distinguish the potential errors in a data set, and efficiently decipher the meager information it harbored. The reappraisal of the mtDNAs with the age of more than 2000 years from Central Asia gave support to the suggestion of extensively (pre)historical gene admixture in this region.

  20. Representativity and reproducibility of DNA malignancy grading in different carcinomas.

    Science.gov (United States)

    Böcking, A; Chatelain, R; Homge, M; Daniel, R; Gillissen, A; Wohltmann, D

    1989-04-01

    The reproducibility of the determination of the "DNA malignancy grade" (DNA-MG) was tested in 56 carcinomas of the colon, breast and lung while its representativity was tested on 195 slides from 65 tumors of the colon, breast and lung. DNA measurements were performed on Feulgen-stained smears with the TAS Plus TV-based image analysis system combined with an automated microscope. The variance of the DNA values of tumor cells around the 2c peak, the "2c deviation index" (2cDI), was taken as a basis for the computation of the DNA-MG, which ranges on a continuous scale from 0.01 to 3.00. The representativity, analyzed by comparison of the DNA-MGs measured in three different areas of the same tumor greater than or equal to 1.5 cm apart from each other, yielded an 81% agreement. No significant differences between DNA-MGs of these areas were found. The intraobserver and interobserver reproducibilities of the DNA grading system, investigated by repeated DNA measurements, were 83.9% and 82.2%, respectively. In comparison, histopathologic grading of the 27 breast cancers studied yielded 65% intraobserver and 57% interobserver reproducibilities and 66% representativity.

  1. Ionic switch controls the DNA state in phage λ.

    Science.gov (United States)

    Li, Dong; Liu, Ting; Zuo, Xiaobing; Li, Tao; Qiu, Xiangyun; Evilevitch, Alex

    2015-07-27

    We have recently found that DNA packaged in phage λ undergoes a disordering transition triggered by temperature, which results in increased genome mobility. This solid-to-fluid like DNA transition markedly increases the number of infectious λ particles facilitating infection. However, the structural transition strongly depends on temperature and ionic conditions in the surrounding medium. Using titration microcalorimetry combined with solution X-ray scattering, we mapped both energetic and structural changes associated with transition of the encapsidated λ-DNA. Packaged DNA needs to reach a critical stress level in order for transition to occur. We varied the stress on DNA in the capsid by changing the temperature, packaged DNA length and ionic conditions. We found striking evidence that the intracapsid DNA transition is 'switched on' at the ionic conditions mimicking those in vivo and also at the physiologic temperature of infection at 37°C. This ion regulated on-off switch of packaged DNA mobility in turn affects viral replication. These results suggest a remarkable adaptation of phage λ to the environment of its host bacteria in the human gut. The metastable DNA state in the capsid provides a new paradigm for the physical evolution of viruses.

  2. Sequence-specific nucleic acid mobility using a reversible block copolymer gel matrix and DNA amphiphiles (lipid-DNA) in capillary and microfluidic electrophoretic separations.

    Science.gov (United States)

    Wagler, Patrick; Minero, Gabriel Antonio S; Tangen, Uwe; de Vries, Jan Willem; Prusty, Deepak; Kwak, Minseok; Herrmann, Andreas; McCaskill, John S

    2015-10-01

    Reversible noncovalent but sequence-dependent attachment of DNA to gels is shown to allow programmable mobility processing of DNA populations. The covalent attachment of DNA oligomers to polyacrylamide gels using acrydite-modified oligonucleotides has enabled sequence-specific mobility assays for DNA in gel electrophoresis: sequences binding to the immobilized DNA are delayed in their migration. Such a system has been used for example to construct complex DNA filters facilitating DNA computations. However, these gels are formed irreversibly and the choice of immobilized sequences is made once off during fabrication. In this work, we demonstrate the reversible self-assembly of gels combined with amphiphilic DNA molecules, which exhibit hydrophobic hydrocarbon chains attached to the nucleobase. This amphiphilic DNA, which we term lipid-DNA, is synthesized in advance and is blended into a block copolymer gel to induce sequence-dependent DNA retention during electrophoresis. Furthermore, we demonstrate and characterize the programmable mobility shift of matching DNA in such reversible gels both in thin films and microchannels using microelectrode arrays. Such sequence selective separation may be employed to select nucleic acid sequences of similar length from a mixture via local electronics, a basic functionality that can be employed in novel electronic chemical cell designs and other DNA information-processing systems. PMID:26095642

  3. Phylogenetic Analysis of Shewanella Strains by DNA Relatedness Derived from Whole Genome Microarray DNA-DNA Hybridization and Comparison with Other Methods

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Liyou; Yi, T. Y.; Van Nostrand, Joy; Zhou, Jizhong

    2010-05-17

    Phylogenetic analyses were done for the Shewanella strains isolated from Baltic Sea (38 strains), US DOE Hanford Uranium bioremediation site [Hanford Reach of the Columbia River (HRCR), 11 strains], Pacific Ocean and Hawaiian sediments (8 strains), and strains from other resources (16 strains) with three out group strains, Rhodopseudomonas palustris, Clostridium cellulolyticum, and Thermoanaerobacter ethanolicus X514, using DNA relatedness derived from WCGA-based DNA-DNA hybridizations, sequence similarities of 16S rRNA gene and gyrB gene, and sequence similarities of 6 loci of Shewanella genome selected from a shared gene list of the Shewanella strains with whole genome sequenced based on the average nucleotide identity of them (ANI). The phylogenetic trees based on 16S rRNA and gyrB gene sequences, and DNA relatedness derived from WCGA hybridizations of the tested Shewanella strains share exactly the same sub-clusters with very few exceptions, in which the strains were basically grouped by species. However, the phylogenetic analysis based on DNA relatedness derived from WCGA hybridizations dramatically increased the differentiation resolution at species and strains level within Shewanella genus. When the tree based on DNA relatedness derived from WCGA hybridizations was compared to the tree based on the combined sequences of the selected functional genes (6 loci), we found that the resolutions of both methods are similar, but the clustering of the tree based on DNA relatedness derived from WMGA hybridizations was clearer. These results indicate that WCGA-based DNA-DNA hybridization is an idea alternative of conventional DNA-DNA hybridization methods and it is superior to the phylogenetics methods based on sequence similarities of single genes. Detailed analysis is being performed for the re-classification of the strains examined.

  4. Searching target sites on DNA by proteins: Role of DNA dynamics under confinement

    Science.gov (United States)

    Mondal, Anupam; Bhattacherjee, Arnab

    2015-01-01

    DNA-binding proteins (DBPs) rapidly search and specifically bind to their target sites on genomic DNA in order to trigger many cellular regulatory processes. It has been suggested that the facilitation of search dynamics is achieved by combining 3D diffusion with one-dimensional sliding and hopping dynamics of interacting proteins. Although, recent studies have advanced the knowledge of molecular determinants that affect one-dimensional search efficiency, the role of DNA molecule is poorly understood. In this study, by using coarse-grained simulations, we propose that dynamics of DNA molecule and its degree of confinement due to cellular crowding concertedly regulate its groove geometry and modulate the inter-communication with DBPs. Under weak confinement, DNA dynamics promotes many short, rotation-decoupled sliding events interspersed by hopping dynamics. While this results in faster 1D diffusion, associated probability of missing targets by jumping over them increases. In contrast, strong confinement favours rotation-coupled sliding to locate targets but lacks structural flexibility to achieve desired specificity. By testing under physiological crowding, our study provides a plausible mechanism on how DNA molecule may help in maintaining an optimal balance between fast hopping and rotation-coupled sliding dynamics, to locate target sites rapidly and form specific complexes precisely. PMID:26400158

  5. DNA Nanotechnology for Cancer Therapy

    OpenAIRE

    Kumar, Vinit; Palazzolo, Stefano; Bayda, Samer; Corona, Giuseppe; Toffoli, Giuseppe; Rizzolio, Flavio

    2016-01-01

    DNA nanotechnology is an emerging and exciting field, and represents a forefront frontier for the biomedical field. The specificity of the interactions between complementary base pairs makes DNA an incredible building material for programmable and very versatile two- and three-dimensional nanostructures called DNA origami. Here, we analyze the DNA origami and DNA-based nanostructures as a drug delivery system. Besides their physical-chemical nature, we dissect the critical factors such as sta...

  6. Local chromatin microenvironment determines DNMT activity : from DNA methyltransferase to DNA demethylase or DNA dehydroxymethylase

    NARCIS (Netherlands)

    van der Wijst, Monique G. P.; Venkiteswaran, Muralidhar; Chen, Hui; Xu, Guo-Liang; Plosch, Torsten; Rots, Marianne G.

    2015-01-01

    Insights on active DNA demethylation disproved the original assumption that DNA methylation is a stable epigenetic modification. Interestingly, mammalian DNA methyltransferases 3A and 3B (DNMT-3A and -3B) have also been reported to induce active DNA demethylation, in addition to their well-known fun

  7. Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase.

    Science.gov (United States)

    Manrao, Elizabeth A; Derrington, Ian M; Laszlo, Andrew H; Langford, Kyle W; Hopper, Matthew K; Gillgren, Nathaniel; Pavlenok, Mikhail; Niederweis, Michael; Gundlach, Jens H

    2012-04-01

    Nanopore technologies are being developed for fast and direct sequencing of single DNA molecules through detection of ionic current modulations as DNA passes through a pore's constriction. Here we demonstrate the ability to resolve changes in current that correspond to a known DNA sequence by combining the high sensitivity of a mutated form of the protein pore Mycobacterium smegmatis porin A (MspA) with phi29 DNA polymerase (DNAP), which controls the rate of DNA translocation through the pore. As phi29 DNAP synthesizes DNA and functions like a motor to pull a single-stranded template through MspA, we observe well-resolved and reproducible ionic current levels with median durations of ∼28 ms and ionic current differences of up to 40 pA. Using six different DNA sequences with readable regions 42-53 nucleotides long, we record current traces that map to the known DNA sequences. With single-nucleotide resolution and DNA translocation control, this system integrates solutions to two long-standing hurdles to nanopore sequencing. PMID:22446694

  8. Curcumin-Mediated HDAC Inhibition Suppresses the DNA Damage Response and Contributes to Increased DNA Damage Sensitivity.

    Directory of Open Access Journals (Sweden)

    Shu-Huei Wang

    Full Text Available Chemo- and radiotherapy cause multiple forms of DNA damage and lead to the death of cancer cells. Inhibitors of the DNA damage response are candidate drugs for use in combination therapies to increase the efficacy of such treatments. In this study, we show that curcumin, a plant polyphenol, sensitizes budding yeast to DNA damage by counteracting the DNA damage response. Following DNA damage, the Mec1-dependent DNA damage checkpoint is inactivated and Rad52 recombinase is degraded by curcumin, which results in deficiencies in double-stand break repair. Additive effects on damage-induced apoptosis and the inhibition of damage-induced autophagy by curcumin were observed. Moreover, rpd3 mutants were found to mimic the curcumin-induced suppression of the DNA damage response. In contrast, hat1 mutants were resistant to DNA damage, and Rad52 degradation was impaired following curcumin treatment. These results indicate that the histone deacetylase inhibitor activity of curcumin is critical to DSB repair and DNA damage sensitivity.

  9. Active DNA demethylation by DNA repair: Facts and uncertainties.

    Science.gov (United States)

    Schuermann, David; Weber, Alain R; Schär, Primo

    2016-08-01

    Pathways that control and modulate DNA methylation patterning in mammalian cells were poor